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pPICZ A, B, and C

Pichia expression vectors for selection on

Zeocin

™

and purification of recombinant proteins

Catalog no. V190-20

Rev. Date: 7 July 2010

Manual part no. 25-0148

MAN00000034

User Manual

Table of Contents

Kit Contents iv

Cloning into pPICZ A, B, 3

Expression 13

.17

Zeocin

™

..................................................................................................................................................................19

Map and Features of pPICZ A, B, 21

Lithium Chloride 23

Construction of In 24

iii

Kit Contents and Storage

Contents

Shipping/Storage

The following components are included with Catalog no. V190–20. Note that the

pPICZ expression vectors are supplied in suspension.

Component Quantity Composition

pPICZ A Expression Vector 20 μg

40 μl of 0.5 μg/μl vector in

10 mM Tris–HCl, 1 mM EDTA,

pH 8.0

pPICZ B Expression Vector 20 μg

40 μl of 0.5 μg/μl vector in

10 mM Tris–HCl, 1 mM EDTA,

pH 8.0

pPICZ C Expression Vector 20 μg

40 μl of 0.5 μg/μl vector in

10 mM Tris–HCl, 1 mM EDTA,

pH 8.0

GS115/pPICZ/lacZ Positive 1 stab --

Control strain

The components included with Catalog no. V190–20 are shipped on wet ice.

Upon receipt, store as directed below.

For long-term storage of your positive control stab strain, we recommend

preparing a glycerol stock immediately upon receipt and storing at –80°C.

Component Shipping Storage

pPICZ A Expression Vector Wet ice Store at –20°C

pPICZ B Expression Vector Wet ice Store at –20°C

pPICZ C Expression Vector Wet ice Store at –20°C

GS115/pPICZ/lacZ positive control strain Wet ice Store at 4°C

Accessory Products

Additional

Products

The products listed in this section are intended for use with the pPICZ vectors.

For more information, visit our web site at or contact

Technical Support (page 32).

Product Quantity Catalog no.

E-Gel

Agarose

Gels

X-33 Pichia strain 1 stab C180-00

GS115 Pichia strain 1 stab C181-00

KM71H Pichia strain 1 stab C182-00

SMD1168H Pichia strain 1 stab C184-00

pPICZα A, B, and C

20 μg each V195-20

pPIC6α A,B, and C

20 μg each V215-20

pPIC6 A, B, and C 20 μg each V210-20

pPIC6 Starter Kit 1 kit K210-01

Original Pichia Expression Kit 1 kit K1710-01

EasySelect

™

Pichia Expression Kit 1 kit K1740-01

Pichia EasyComp

™

Transformation Kit 1 kit K1730-01

Pichia Protocols 1 book G100-01

PureLink

™

Gel Extraction Kit 50 preps K2100–12

250 preps K2100–25

S.N.A.P

™

Gel Purification Kit 25 preps K1999–25

PureLink

™

Quick Plasmid Miniprep Kit 50 preps K2100–10

250 preps K2100–11

PureLink

™

HiPure Plasmid Midiprep Kit 25 preps K2100–04

50 preps K2100–13

One Shot

TOP10 (chemically competent E. coli) 10 reactions C4040–10

20 reactions C4040–03

One Shot

TOP10 Electrocompetent E. Coli 10 reactions C4040-50

20 reactions C4040-52

TOP10 Electrocomp

™

Kits 20 reactions C664–55

Positope

™

Control Protein 5 μg R900-50

CIAP (Calf Intestinal Alkaline Phosphatase) 1,000 units 18009–019

T4 DNA Ligase 100 units 15224–017

500 units 15224–025

Zeocin

™

1 g R250-01

5 g R250-05

β-Gal Assay Kit

1 kit K1455-01

β-Gal Staining Kit

1 kit K1465-01

E-Gel

Agarose Gels are bufferless, pre-cast agarose gels designed for fast,

convenient electrophoresis of DNA samples. E-Gel

agarose gels are available in

different agarose percentage and well format for your convenience.

For more details on these products, visit our web site at or

contact Technical Support (page 32).

Continued on next page

Accessory Products,

Continued

Zeocin

™

Detection of

Fusion Protein

Purification of

Fusion Protein

Zeocin

™

may be obtained from Invitrogen (see above). For your convenience, the

drug is prepared in autoclaved, deionized water and available in 1.25 ml aliquots

at a concentration of 100 mg/ml. The stability of Zeocin

™

is guaranteed for six

months if stored at –20°C.

A number of antibodies are available from Invitrogen to detect expression of

your fusion protein from the pPICZ vector. Horseradish peroxidase (HRP)-

conjugated antibodies allow one-step detection in Western blots using

colorimetric or chemiluminescent detection methods. The amount of antibody

supplied is sufficient for 25 Western Blots.

Antibody Epitope Catalog no.

Anti-myc R950–25

Detects the 10 amino acid epitope

Anti-myc-HRP

derived from c-myc (Evans et al., 1985):

R951–25

EQKLISEEDL

Anti-His(C-term) R930–25

Detects the C-terminal polyhistidine

Anti-His(C-term)-HRP

(6xHis) tag (requires the free carboxyl

group for detection) (Lindner et al., 1997):

R931–25

HHHHHH-COOH

The polyhistidine (6xHis) tag allows purification of the recombinant fusion

protein using metal-chelating resins such as ProBond

™

. Ordering information for

ProBond

™

resin is provided below.

Product Quantity Catalog no.

ProBond

™

Purification System 6 purifications K850–01

ProBond

™

Purification System with Anti-myc-1 Kit K852–01

HRP Antibody

ProBond

™

Purification System with Anti-1 Kit K853–01

His(C-term)-HRP Antibody

ProBond

™

Nickel-Chelating Resin 50 ml R801–01

150 ml R801–15

Purification Columns 50 each R640–50

Introduction

Overview

Introduction

Reference

Sources

Recommended

Pichia Host Strain

pPICZ A, B, and C are 3.3 kb expression vectors used to express recombinant

proteins in Pichia pastoris. Recombinant proteins are expressed as fusions to a

C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.

The vector allows high-level, methanol inducible expression of the gene of

interest in Pichia, and can be used in any Pichia strain including X33, GS115,

SMD1168H, and KM71H. pPICZ contains the following elements:

• 5′ fragment containing the AOX1 promoter for tightly regulated, methanol-

induced expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989;

Tschopp et al., 1987a)

• Zeocin

™

resistance gene for selection in both E. coli and Pichia (Baron et al.,

1992; Drocourt et al., 1990)

• C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis)

tag for detection and purification of a recombinant fusion protein (if desired)

• Three reading frames to facilitate in-frame cloning with the C-terminal

peptide

The pPICZ A, B, and C expression vectors may be used with the Original Pichia

Expression Kit, and are included in the EasySelect

™

Pichia Expression Kit (see

page v for ordering information). Additional general information about

recombinant protein expression in Pichia pastoris is provided in the manuals for

the Original Pichia Expression Kit and the EasySelect

™

Pichia Expression Kit. For

more information about the Original Pichia Expression Kit, the EasySelect

™

Pichia

Expression Kit, or their manuals, visit our web site at or

contact Technical Support (page 32).

More detailed information and protocols dealing with

Pichia pastoris may also be

found in the following general reference:

Higgins, D. R., and Cregg, J. M. (1998) Pichia Protocols. In Methods in Molecular

Biology, Vol. 103. (J. M. Walker, ed. Humana Press, Totowa, NJ) (see page v for

ordering information).

We recommend using the X-33 Pichia strain as the host for expression of

recombinant proteins from pPICZ. Other Pichia strains including GS115, KM71H,

and SMD1168H are suitable. The X-33 Pichia strain and other strains are

available from Invitrogen (see page v for ordering information). The X-33 Pichia

strain has the following genotype and phenotype:

Genotype: Wild-type

Phenotype: Mut

Overview,

Continued

Experimental

Overview

The following table describes the basic steps needed to clone and express your

gene of interest in pPICZ.

Step Action

1 Propagate pPICZ A, B, and C by transformation into a recA, endA1

E. coli strain such as TOP10, DH5 , or JM109.

2 Develop a cloning strategy and ligate your gene into one of the pPICZ

vectors in frame with the C-terminal tag.

3 Transform into E. coli and select transformants on Low Salt LB plates

containing 25

g/ml Zeocin

™

4 Analyze 10–20 transformants by restriction mapping or sequencing to

confirm in-frame fusion of your gene with the C-terminal tag.

5 Purify and linearize the recombinant plasmid for transformation into

Pichia pastoris.

6 Transform your Pichia strain and plate onto YPDS plates containing the

appropriate concentration of Zeocin

™

7 Select for Zeocin

™

-resistant transformants.

8 Optimize expression of your gene.

9 Purify your fusion protein on metal-chelating resin (i.e. ProBond

™

Continued on next page

Methods

Cloning into pPICZ A, B, and C

Introduction

General Molecular

Biology

Techniques

E. coli Strain

Transformation

Method

Maintenance of

Plasmids

The pPICZ vector is supplied with the multiple cloning site in three reading

frames (A, B, and C) to facilitate cloning your gene of interest in frame with the

C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.

Use the diagrams provided on pages 5–7 to help you design a strategy to clone

your gene of interest in frame with the C-terminal peptide. General

considerations for cloning and transformation are discussed in this section.

For assistance with E. coli transformations, restriction enzyme analysis, DNA

biochemistry, and plasmid preparation, refer to Molecular Cloning: A Laboratory

Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel

et al., 1994).

Many E. coli strains are suitable for the propagation of the pPICZ vectors

including TOP10, JM109, and DH5 . We recommend that you propagate the

pPICZ vectors in E. coli strains that are recombination deficient (recA) and

endonuclease A deficient (endA).

For your convenience, TOP10 E. coli are available as chemically competent or

electrocompetent cells from Invitrogen (page v).

You may use any method of choice for transformation. Chemical transformation

is the most convenient for many researchers. Electroporation is the most efficient

and the method of choice for large plasmids.

The pPICZ vectors contain the Zeocin

™

resistance (Sh ble) gene to allow selection

of the plasmid using Zeocin

™

. To propagate and maintain the pPICZ plasmids,

we recommend using the following procedure:

1. Use 10 ng of your vector to transform a recA, endA E. coli strain like TOP10,

DH5 , JM109, or equivalent (see above).

2. Select transformants on Low Salt LB plates containing 25

g/ml Zeocin

™

(see

page 17 for a recipe).

3. Prepare a glycerol stock from each transformant containing plasmid for

long-term storage (see page 8).

Continued on next page

Cloning into pPICZ A, B, and C,

Continued

General

Considerations

Cloning

Considerations

Construction of

Multimeric

Plasmids

The following are some general points to consider when using pPICZ to express

your gene of interest in Pichia:

• The codon usage in Pichia is believed to be similar to Saccharomyces cerevisiae.

• Many Saccharomyces genes have proven to be functional in Pichia.

• The premature termination of transcripts because of "AT rich regions" has

been observed in Pichia and other eukaryotic systems (Henikoff & Cohen,

1984; Irniger et al., 1991; Scorer et al., 1993; Zaret & Sherman, 1984). If you

have problems expressing your gene, check for premature termination by

northern analysis and check your sequence for AT rich regions. It may be

necessary to change the sequence in order to express your gene (Scorer et al.,

1993).

• The native 5´ end of the AOX1 mRNA is noted in the diagram for each

multiple cloning site. This information is needed to calculate the size of the

expressed mRNA of the gene of interest if you need to analyze mRNA for

any reason.

For proper initiation of translation, your insert should contain an initiation ATG

codon as part of a yeast consensus sequence (Romanos et al., 1992). An example

of a yeast consensus sequence is provided below. The ATG initiation codon is

shown underlined.

(G/A)NNATG

To express your gene as a recombinant fusion protein, you must clone your gene

in frame with the C-terminal peptide containing the c-myc epitope and the

polyhistidine tag. The vector is supplied in three reading frames to facilitate

cloning. Refer to the diagrams on pages 5–7 to develop a cloning strategy.

If you wish to express your protein without the C-terminal peptide, be sure to

include a stop codon.

pPICZ A, B, and C contain unique Bgl II and BamH I sites to allow construction

of plasmids containing multiple copies of your gene. For information on how to

construct multimers, refer to pages 24–31.

Continued on next page

Cloning into pPICZ A, B, and C,

Continued

Multiple Cloning

Site of pPICZ A

Below is the multiple cloning site for pPICZ A. Restriction sites are labeled to

indicate the cleavage site. The boxed nucleotides indicate the variable region.

The multiple cloning site has been confirmed by sequencing and functional

testing.

You can download the complete sequence of pPICZ A from our web site at

or by contacting

Technical Support (see page 32).

For a map and a description of the features of pPICZ, refer to the Appendix

(pages 21–22).

5´ end of AOX1 mRNA

5´ AOX1 priming site

811AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA

871

931

CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT

Sfu IEcoR IPml ISfi IBsmB IAsp718 IKpn IXho I

ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC

Sac IINot IApa I

myc epitope

991GGCGGCCGCC AGCTT GGGCCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

Polyhistidine tag

1042AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CATTGA GTTTTAGCCT TAGACATGAC

Asn Ser Ala Val Asp His His His His His His ***

TGTTCCTCAG TTCAAGTTGG GCACTTACGA GAAGACCGGT CTTGCTAGAT TCTAATCAAG

3´ AOX1 priming site

1098

1158AGGATGTCAG AATGCCATTT GCCTGAGAGA TGCAGGCTTC ATTTTTGATA CTTTTTTATT

3´polyadenylation site

1218TGTAACCTAT ATAGTATAGG ATTTTTTTTG TCATTTTGTT

Continued on next page

Cloning into pPICZ A, B, and C,

Continued

Multiple Cloning

Site of pPICZ B

Below is the multiple cloning site for pPICZ B. Restriction sites are labeled to

indicate the cleavage site. The boxed nucleotides indicate the variable region.

The multiple cloning site has been confirmed by sequencing and functional

testing.

You can download the complete sequence of pPICZ B from our web site at

or by contacting

Technical Support (see page 32).

For a map and a description of the features of pPICZ, refer to the Appendix

(pages 21–22).

5´ end of AOX1 mRNA

5´ AOX1 priming site

811AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA

871CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT

Sfu IEcoR IPml ISfi IBsmB IAsp718 IKpn IXho I

931

991

ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC

Sac IINot IXba I

myc epitope

GGCGGCCGCC AGCTT TCTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

Polyhistidine tag

1040

1096

1156

1216

AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGATGA GTTTGTAGCC TTAGACATGA

Asn Ser Ala Val Asp His His His His His His ***

CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA

3´ AOX1 priming site

GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT

3´ polyadenylation site

TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC

Continued on next page

Cloning into pPICZ A, B, and C,

Continued

Multiple Cloning

Site of pPICZ C

Below is the multiple cloning site for pPICZ C. Restriction sites are labeled to

indicate the cleavage site. The boxed nucleotides indicate the variable region.

The multiple cloning site has been confirmed by sequencing and functional

testing.

You can download the complete sequence of pPICZ C from our web site at

or by contacting

Technical Support (see page 32).

For a map and a description of the features of pPICZ, refer to the Appendix

(pages 21–22).

5´ end of AOX1 mRNA

5´ AOX1 priming site

811AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA

871

931

991

CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT

Sfu IEcoR IPml ISfi IBsmB IAsp718 IKpn IXho I

ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC

Sac IINot ISnaB I

myc epitope

GGCGGCCGCC AGCTT ACGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

Polyhistidine tag

1041

1097

AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGATGA GTTTGTAGCC TTAGACATGA

Asn Ser Ala Val Asp His His His His His His ***

CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA

3´ AOX1 priming site

1157

1217

GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT

3´ polyadenylation site

TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC

Continued on next page

Cloning into pPICZ A, B, and C,

Continued

E. coli

Transformation

Important

Preparing a

Glycerol Stock

Plasmid

Preparation

Transform your ligation mixtures into a competent recA, endA E. coli strain

(e.g. TOP10, DH5

, JM109) and select on Low Salt LB agar plates containing

g/ml Zeocin

™

(see below). Note that there is no blue/white screening for the

presence of insert with pPICZ A, B, or C. Once you have obtained Zeocin

™

resistant colonies, pick 10 transformants and screen for the presence and

orientation of your insert.

To facilitate selection of Zeocin

™

-resistant E. coli, the salt concentration of the

medium must remain low (<90 mM) and the pH must be 7.5. Prepare Low Salt

LB broth and plates using the recipe in the Appendix, page 17.

Failure to lower the salt content of your LB medium will result in non-

selection due to inhibition of the drug.

We recommend that you sequence your construct to confirm that your gene is in

the correct orientation for expression and cloned in frame with the C-terminal

peptide (if desired). Refer to the diagrams on pages 5–7 for the sequences and

location of the priming sites.

Once you have identified the correct clone, be sure to purify the colony and make

a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock

of your plasmid at –20°C.

1. Streak the original colony out on an Low Salt LB plate containing 25

g/ml

Zeocin

™

. Incubate the plate at 37°C overnight.

2. Isolate a single colony and inoculate into 1–2 ml of Low Salt LB containing

g/ml Zeocin

™

3. Grow the culture to mid-log phase (OD

600

= 0.5–0.7).

4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a

cryovial.

5. Store at –80°C.

Once you have cloned and sequenced your insert, generate enough plasmid

DNA to transform Pichia (5–10

g of each plasmid per transformation). We

recommend isolating plasmid DNA using the PureLink

™

Quick Plasmid

Miniprep Kit or the PureLink

™

HiPure Plasmid Midiprep Kit (page v), or CsCl

gradient centrifugation.

Once you have purified plasmid DNA, proceed to

Pichia Transformation, next

page.

Pichia Transformation

Introduction

You should now have your gene cloned into one of the pPICZ vectors. Your

construct should be correctly fused to the C-terminal peptide (if desired). This

section provides general guidelines to prepare plasmid DNA, transform your

Pichia strain, and select for Zeocin

™

-resistant clones.

We generally use 100

g/ml Zeocin

™

to select for transformants when using the

X-33 Pichia strain. If you are transforming your pPICZ construct into another

Pichia strain, note that selection conditions may vary. We recommend

performing a dose response curve to determine the appropriate concentration of

Zeocin

™

to use for selection of transformants in your strain.

We do not recommend spheroplasting for transformation of Pichia with

plasmids containing the Zeocin

™

resistance marker. Spheroplasting involves

removal of the cell wall to allow DNA to enter the cell. Cells must first

regenerate the cell wall before they are able to express the Zeocin

™

resistance

gene. For this reason, plating spheroplasts directly onto selective medium

containing Zeocin

™

does not yield any transformants.

We recommend electroporation for transformation of Pichia with pPICZ A, B,

or C. Electroporation yields 10

to 10

transformants per

g of linearized DNA

and does not destroy the cell wall of Pichia. If you do not have access to an

electroporation device, use the LiCl protocol on page 23 or the Pichia EasyComp

™

Transformation Kit available from Invitrogen (see below).

If you wish to perform chemical transformation of your Pichia strain with pPICZ

Pichia

™

A, B, or C, the Pichia EasyComp

™

Transformation Kit is available from Invitrogen

EasyComp

Transformation Kit

(see page v for ordering information). The Pichia EasyComp

™

Transformation Kit

provides reagents to prepare 6 preparations of competent cells. Each preparation

will yield enough competent cells for 20 transformations. Competent cells may

be used immediately or frozen and stored for future use. For more information,

visit our web site at or contact

Technical Support (page

32).

Since pPICZ does not contain the HIS4 gene, integration can only occur at the

AOX1 locus. Vector linearized within the 5´ AOX1 region will integrate by gene

insertion into the host 5´ AOX1 region. Therefore, the Pichia host that you use

will determine whether the recombinant strain is able to metabolize methanol

(Mut

) or not (Mut

). To generate a Mut

recombinant strain, you must use a

Pichia host that contains the native AOX1 gene (e.g. X-33, GS115, SMD1168H). If

you wish to generate a Mut

recombinant strain, then use a Pichia host that has a

disrupted AOX1 gene (i.e. KM71H).

Continued on next page

Zeocin Selection

™

Method of

Transformation

Important

Pichia Transformation,

Continued

His4 Host Strains

Materials Needed

Linearizing Your

pPICZ Construct

Restriction Digest

Host strains containing the his4 allele (e.g. GS115) and transformed with the

pPICZ vectors require histidine when grown in minimal media. Add histidine to

a final concentration of 0.004% to ensure growth of your transformants.

The pPICZ vectors do not contain a yeast origin of replication. Transformants

can only be isolated if recombination occurs between the plasmid and the Pichia

genome.

You will need the following items:

Note: Inclusion of sorbitol in YPD plates stabilizes electroporated cells as they appear to

be somewhat osmotically sensitive.

• 5–10

g pure pPICZ containing your insert

• YPD Medium

• 50 ml conical polypropylene tubes

• 1 liter cold (4°C) sterile water (place on ice the day of the experiment)

• 25 ml cold (4°C) sterile 1 M sorbitol (place on ice the day of the experiment)

• 30°C incubator

• Electroporation device and 0.2 cm cuvettes

• YPDS plates containing the appropriate concentration of Zeocin

™

(see

page 18 for recipe)

To promote integration, we recommend that you linearize your pPICZ construct

within the 5′ AOX1 region. The table below lists unique sites that may be used to

linearize pPICZ prior to transformation. Other restriction sites are possible.

Note that for the enzymes listed below, the cleavage site is the same for versions

A, B, and C of pPICZ. Be sure that your insert does not contain the restriction site

you wish to use to linearize your vector.

Enzyme Restriction Site (bp) Supplier

Sac I 209 Many

Pme I 414 New England Biolabs

BstX I 707 Many

1. Digest ~5–10

g of plasmid DNA with one of the enzymes listed above.

2. Check a small aliquot of your digest by agarose gel electrophoresis for

complete linearization.

3. If the vector is completely linearized, heat inactivate or add EDTA to stop

the reaction, phenol/chloroform extract once, and ethanol precipitate using

1/10 volume 3 M sodium acetate and 2.5 volumes of 100% ethanol.

4. Centrifuge the solution to pellet the DNA, wash the pellet with 80% ethanol,

air-dry, and resuspend in 10

l sterile, deionized water. Use immediately or

store at –20°C.

Continued on next page

Pichia Transformation,

Continued

Preparation of

Pichia for

Electroporation

Transformation by

Electroporation

Follow the procedure below to prepare your Pichia pastoris strain for

electroporation.

1. Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical tube at

30°C overnight.

2. Inoculate 500 ml of fresh medium in a 2 liter flask with 0.1–0.5 ml of the

overnight culture. Grow overnight again to an OD

600

= 1.3–1.5.

3. Centrifuge the cells at 1500 × g for 5 minutes at 4°C. Resuspend the pellet

with 500 ml of ice-cold (0–4°C), sterile water.

4. Centrifuge the cells as in Step 3, then resuspend the pellet with 250 ml of

ice-cold (0–4°C), sterile water.

5. Centrifuge the cells as in Step 3, then resuspend the pellet in 20 ml of ice-

cold (0–4°C) 1 M sorbitol.

6. Centrifuge the cells as in Step 3, then resuspend the pellet in 1 ml of ice-cold

(0–4°C) 1 M sorbitol for a final volume of approximately 1.5 ml. Keep the

cells on ice and use that day. Do not store cells.

1. Mix 80

l of the cells from Step 6 (above) with 5–10

g of linearized pPICZ

DNA (in 5–10

l sterile water) and transfer them to an ice-cold (0–4°C)

0.2 cm electroporation cuvette.

2. Incubate the cuvette with the cells on ice for 5 minutes.

3. Pulse the cells according to the parameters for yeast (Saccharomyces

cerevisiae) as suggested by the manufacturer of the specific electroporation

device being used.

4. Immediately add 1 ml of ice-cold 1 M sorbitol to the cuvette. Transfer the

cuvette contents to a sterile 15 ml tube.

5. Let the tube incubate at 30°C without shaking for 1 to 2 hours.

6. Spread 50-200

l each on separate, labeled YPDS plates containing the

appropriate concentration of Zeocin

™

7. Incubate plates for 2–3 days at 30°C until colonies form.

8. Pick 10–20 colonies and purify (streak for single colonies) on fresh YPD or

YPDS plates containing the appropriate concentration of Zeocin

™

Continued on next page

Pichia Transformation,

Continued

Mut Phenotype

Generally, several hundred Zeocin

™

-resistant colonies are generated using the

protocol on the previous page. If more colonies are needed, the protocol may be

modified as described below. Note that you will need ~20, 150 mm plates with

YPDS agar containing the appropriate concentration of Zeocin

™

1. Set up two transformations per construct and follow Steps 1 through 5 of

the Transformation by Electroporation protocol, page 11.

After 1 hour in 1 M sorbitol at 30°C (Step 5, previous page), add 1 ml YPD

medium to each tube.

3. Shake (~200 rpm) the cultures at 30°C.

4. After 1 hour, take one of the tubes and plate out all of the cells by spreading

200

l on 150 mm plates containing the appropriate concentration of

Zeocin

™

5. Optional: Continue incubating the other culture for three more hours (for a

total of four hours) and then plate out all of the cells by spreading 200

l on

150 mm plates containing the appropriate concentration of Zeocin

™

6. Incubate plates for 2–4 days at 30°C until colonies form.

If you used a Pichia strain containing a native AOX1 gene (e.g. X-33, GS115,

SMD1168H) as the host for your pPICZ construct, your Zeocin

™

-resistant

transformants will be Mut

. If you used a strain containing a deletion in the

AOX1 gene (e.g. KM71H), your transformants will be Mut

If you wish to verify the Mut phenotype of your Zeocin

™

-resistant transformants,

you may refer to the general guidelines provided in the EasySelect

™

Pichia

Expression Kit manual or the Original Pichia Expression Kit manual or to

published reference sources (Higgins & Cregg, 1998).

You are now ready to test your transformants for expression of your gene of

interest. See Expression in Pichia, next page.

Expression in Pichia

Introduction

The primary purpose of small-scale expression is to identify/confirm a

recombinant Pichia clone that is expressing the correct protein. Small-scale

expression conditions may not be optimal for your protein. For this reason, the

method you choose for detection (e.g. SDS-PAGE, Western, or functional assay)

may be an important factor in determining the success of expression. If your

method of detection does not reveal any expression, you may want to consider

using a more sensitive method.

Once a positive clone has been identified, large-scale expression can be carried

out in shake flask or fermentation, and expression conditions can be optimized.

Control Strain

As a positive control for expression, GS115/pPICZ/lacZ is provided. For

expression, use the small-scale Mut

protocol described in the Pichia Expression

System manual. Expression in shake flasks is detectable after 48 hours and

reaches the maximum at 96 hours (4 days). β-galactosidase is detected using

SDS-PAGE and staining the gel with Coomassie Blue or the ONPG assay (

-Gal

Assay page v). Cells expressing

β-galactosidase

can be detected by plating on

medium containing methanol and X-gal.

Note that once you have obtained Zeocin

™

-resistant transformants, it is not

necessary to maintain your recombinant Pichia clone in medium containing

Zeocin

™

for expression studies. Zeocin

™

is only required for initial screening and

selection of recombinant clones.

We recommend that you use the following techniques to assay expression of

your protein. The C-terminal tag will add 2.5 kDa to the size of your protein. Be

sure to account for any additional amino acids that are in between the end of

your protein and the C-terminal tag.

Method of Detection

Visualization by eye

Antibody to your particular protein

Anti-myc antibodies (see the next page)

Anti-His(C-term) antibodies (see the next

page)

Varies depending on assay.

Sensitivity

Can detect as little as 100 ng in

a single band

Can detect as little as 2 ng in a

single band

Can detect as little as 1–10 pg,

depending on detection

method (alkaline phosphatase,

horseradish peroxidase,

radiolabeled antibody)

Varies depending on assay

Used to compare relative

amounts of protein.

Continued on next page

Detection of

Recombinant

Proteins in Pichia

Technique

SDS-PAGE

(Coomassie-stained)

SDS-PAGE

(Silver-stained)

Western Analysis

Functional assay

Expression in Pichia,

Continued

Polyacrylamide

Gel

Electrophoresis

Western Analysis

Important

Expression

Guidelines

To facilitate separation and visualization of your recombinant protein by

polyacrylamide gel electrophoresis, a wide range of pre-cast NuPAGE

and Tris-

Glycine polyacrylamide gels are available from Invitrogen. The NuPAGE

Gel

System avoids the protein modifications associated with Laemmli-type SDS-

PAGE, ensuring optimal separation for protein analysis. In addition, Invitrogen

also carries a large selection of molecular weight protein standards and staining

kits. For more information about the appropriate gels, standards, and stains to

use to visualize your recombinant protein, visit our web site at

or contact

Technical Support (page 32).

To detect expression of your recombinant fusion protein by Western blot

analysis, you may use the Anti-myc antibodies or the Anti-His(C-term)

antibodies available from Invitrogen (see page × for ordering information) or an

antibody to your protein of interest. In addition, the Positope

™

Control Protein

(page v) is available from Invitrogen for use as a positive control for detection of

fusion proteins containing a c-myc epitope or a polyhistidine (6xHis) tag.

WesternBreeze

™

Chromogenic Kits and WesternBreeze

™

Chemiluminescent Kits

are available from Invitrogen to facilitate detection of antibodies by colorimetric

or chemiluminescent methods. For more information, visit our web site at

or contact

Technical Support (page 32).

Because the pPICZ vector does not contain the HIS4 gene, his4 Pichia strains

containing the integrated plasmid must be grown in medium containing 0.004%

histidine. If histidine is not present in the medium the cells will not grow. If you

use X-33, SMD1168H, or KM71H as the host strain, supplementation of the

medium with histidine is not required.

General guidelines to perform small-scale expression, optimize expression, and

scale-up of expression are provided in the EasySelect

™

Pichia Expression Kit

manual or the Original Pichia Expression Kit manual.

Purification

Introduction

In this section, you will grow and induce a 10–200 ml culture of your Pichia

transformant for trial purification on a metal-chelating resin such as ProBond

™

(page vi). You may harvest the cells and store them at –80°C until you are ready to

purify your fusion protein, or you may proceed directly with protein purification.

Note: This section only describes preparation of cell lysates and sample application onto

ProBond

™

. For instructions on how to prepare and use ProBond

™

resin, refer to the

ProBond

™

Purification System manual.

ProBond Resin

™

We recommend that you use the ProBond

™

Purification System (page vi) to purify

fusion proteins expressed from pPICZ A, B, or C. The ProBond

™

Purification kit

contains six 2 ml precharged, prepacked ProBond

™

resin columns, buffers for

native and denaturing purification, and an instruction manual.

Note: Instructions for equilibration of and chromatography on ProBond

™

resin are

contained in the ProBond

™

Purification Kit.

If you are using a metal-chelating resin other than ProBond

™

, follow the

manufacturer's recommendations to purify fusion proteins expressed in bacteria

or yeast.

Binding Capacity

of ProBond

™

One milliliter of ProBond

™

resin binds at least 1 mg of recombinant protein. This

amount can vary depending on the protein.

Throughout the following protocol, be sure to keep the cell lysate and fractions on

ice. Small-scale purifications using the 2 ml ProBond

™

columns and buffers can be

performed at room temperature on the bench top. For large scale purifications, all

reagents must be kept at 4°C.

Important

Preparation of Cell

Express your protein using a small-scale culture (10–20 ml for Mut

strains;

100–200 ml for Mut

) and the optimal conditions for expression (if determined).

Lysates

Refer to the Pichia Expression Kit manual for details. Once your protein is

expressed, follow the protocol below to prepare a cell lysate for chromatography

on ProBond

™

Prepare Breaking Buffer (BB) as described in the Appendix, page 18.

1. Wash cells once in BB by resuspending them and centrifuging 5–10 minutes

at 3000 × g at 4°C.

2. Resuspend the cells to an OD

600

of 50–100 in BB.

3. Add an equal volume of acid-washed glass beads (0.5 mm). Estimate volume

by displacement.

4. Vortex the mixture for 30 seconds, then incubate on ice for 30 seconds. Repeat

7 more times. Alternating vortexing with cooling keeps the cell extracts cold

and reduces denaturation of your protein.

5. Centrifuge the sample at 4°C for 5–10 minutes at 12,000 × g.

6. Transfer the clear supernatant to a fresh container and analyze for your

protein. The total protein concentration should be around 2–3 mg/ml.

7. Save the pellet and extract with 6 M urea or 1% Triton X-100 to check for

insoluble protein.

Continued on next page

Purification,

Continued

Sample

Application

(Native

Conditions)

Sample

Application

(Denaturing

Conditions)

Analysis of

Purification

Scale-up

For sample application onto ProBond

™

, you will need Native Binding Buffer,

pH 7.8 and a 2 ml ProBond

™

column, pre-equilibrated using native conditions.

1. Combine 1 ml (2–3 mg/ml total protein) of Pichia lysate with 7 ml Native

Binding Buffer.

2. Take a pre-equilibrated ProBond

™

column and resuspend the resin in 4 ml

of the diluted lysate from Step 1.

3. Seal the column and batch-bind by rocking gently at room temperature for

10 minutes.

4. Let the resin settle by gravity or low speed centrifugation (800 × g) and

carefully remove the supernatant. Save the supernatant to check for

unbound protein.

5. Repeat Steps 2 through 4 with the remaining 4 ml of diluted lysate. Proceed

to Column Washing and Elution Under Native Conditions in the

ProBond

™

Purification manual. Use the recommendations noted for

bacterial cell lysates.

Use the protocol above except pre-equilibrate the ProBond

™

column using

Denaturing Binding Buffer and combine 1 ml of the Pichia cell lysate with 7 ml of

the Denaturing Binding Buffer.

We have observed that some Pichia proteins may be retained on the ProBond

™

column using native purification conditions. Optimization of the purification

(see ProBond

™

Purification manual) or using denaturing purification may

remove these non-specific Pichia proteins.

Be sure to save all fractions, washes, and flow-through for analysis by SDS-

PAGE. You may need to use Western blot analysis to detect your protein if

expression is low or not enough protein was loaded onto the column. Refer to

the ProBond

™

Purification System manual for a guide to troubleshoot

chromatography.

You may find it necessary to scale-up your purification to obtain sufficient

amounts of purified protein. Adjust the pH and NaCl concentration of your

lysate with 1/10 volume of 10X Stock Solution B (ProBond

™

Purification Kit)

before adding it to the column. The pH should be greater than or equal to 7.5

and the NaCl concentration should be ~500 mM. Using 10X Stock Solution B to

adjust the pH and the ionic strength keeps the total volume small for sample

application.

Appendix

Recipes

Low Salt LB

Medium with

Zeocin™

YPD (+ Zeocin

™

)

10 g Tryptone

5 g NaCl

5 g Yeast Extract

1. Combine the dry reagents above and add deionized, distilled water to

950 ml. Adjust pH to 7.5 with 1N NaOH. Bring the volume up to 1 liter. For

plates, add 15 g/L agar before autoclaving.

2. Autoclave on liquid cycle at 15 psi and 121°C for 20 minutes.

3. Allow the medium to cool to at least 55°C before adding the Zeocin

™

25 μg/ml final concentration.

4. Store plates at 4°C in the dark. Plates containing Zeocin

™

are stable for up to

2 weeks.

Yeast Extract Peptone Dextrose Medium

(1 liter)

1% yeast extract

2% peptone

Sterile water

2% agar (Optional: If making YPD slants or plates)

2% dextrose (glucose)

Zeocin

™

(in appropriate concentration)

1. Dissolve 10 g 1% yeast extract and 20 g 2% peptone in 900 ml water

2. Optional: Add 20 g of 2% agar if making YPD slants or plates. Dissolve.

3. Autoclave for 20 minutes on liquid cycle.

4. Add 100 ml of 2% dextrose (filter-sterilize dextrose before use).

5. Cool solution to ~60°C and add the appropriate amount of Zeocin

™

from a

100 mg/ml stock solution.

Note: It is necessary to include Zeocin

™

in the medium for selection of Pichia

transformants only. Zeocin

™

may be omitted from the medium when performing

expression studies.

5. Store YPD slants or plates containing Zeocin

™

at 4°C. The shelf life is

1–2 weeks.

Continued on next page

Recipes,

Continued

YPDS + Zeocin

™

Agar

Breaking Buffer

Yeast Extract Peptone Dextrose Medium with Sorbitol

(1 liter)

1% yeast extract

2% peptone

1 M sorbitol

2% agar

Sterile water

2% dextrose (glucose)

Zeocin

™

(in appropriate concentration)

1. Dissolve the following item in 900 ml water:

• 10 g yeast extract

• 182.2 g sorbitol

• 20 g of peptone

2. Add 20 g of 2% agar to the solution and dissolve.

3. Autoclave for 20 minutes on liquid cycle.

4. Add 100 ml of 2% dextrose (filter-sterilize dextrose before use).

5. Cool solution to ~60°C and add the appropriate amount of Zeocin

™

from a

100 mg/ml stock solution. Note: It is necessary to include Zeocin

™

in the

medium for selection of Pichia transformants only. Zeocin

™

may be omitted

from the medium when performing expression studies.

6. Store YPDS slants or plates containing Zeocin

™

at 4°C. The shelf life is one to

two weeks.

50 mM sodium phosphate, pH 7.4

1 mM EDTA

5% glycerol

Sterile water

1 mM PMSF (phenylmethylsulfonyl fluoride. You may use other protease

inhibitors)

1. Prepare a stock solution of your desired protease inhibitors and store

appropriately. Follow manufacturer’s recommendations.

2. For 1 liter, dissolve the following into 900 ml water:

• 6 g sodium phosphate (monobasic)

• 372 mg EDTA

• 50 ml glycerol

3. Use NaOH to adjust pH and bring up the volume to 1 liter. Store at 4°C.

4. Add 1 mM PMSF or other protease inhibitors immediately before use.

Zeocin

™

Zeocin

™

Molecular Weight,

Formula, and

Structure

Applications of

Zeocin™

Zeocin

™

is a member of the bleomycin/phleomycin family of antibiotics isolated

from Streptomyces. Antibiotics in this family are broad spectrum antibiotics that

act as strong anti-bacterial and anti-tumor drugs. They show strong toxicity

against bacteria, fungi (including yeast), plants, and mammalian cells (Baron

et al., 1992; Drocourt et al., 1990; Mulsant et al., 1988; Perez et al., 1989).

The Zeocin

™

resistance protein has been isolated and characterized (Calmels

et al., 1991; Drocourt et al., 1990). This protein, the product of the Sh ble gene

(Streptoalloteichus hindustanus bleomycin gene), is a 13.7 kDa protein that binds

Zeocin

™

and inhibits its DNA strand cleavage activity. Expression of this protein

in eukaryotic and prokaryotic hosts confers resistance to Zeocin

™

The formula for Zeocin

™

is C

and the molecular weight is 1,535. The

diagram below shows the structure of Zeocin

™

CONH

R =

MW = 1,535

Zeocin

™

is used for selection in mammalian cells (Mulsant et al., 1988); plants

(Perez et al., 1989); yeast (Baron et al., 1992); and prokaryotes (Drocourt et al.,

1990). Suggested concentrations of Zeocin

™

for selection in Pichia and E. coli are

listed below:

Organism Zeocin

™

Concentration and Selective Medium

E. coli

25–50

g/ml in Low Salt LB medium

(see page 17 for a recipe)

Pichia

100–1000

g/ml (varies with strain and medium)

Efficient selection requires that the concentration of NaCl be no more than 5 g/L (< 90 mM).

Continued on next page

Zeocin

™

Continued

Handling Zeocin

™

• High salt and acidity or basicity inactivate Zeocin

™

; therefore, we

recommend that you reduce the salt in bacterial medium and adjust the pH

to 7.5 to keep the drug active (see Low Salt LB Medium, page 17). Note that

the salt concentration and pH do not need to be adjusted when preparing

tissue culture medium containing Zeocin

™

• Store Zeocin

™

at –20°C and thaw on ice before use.

• Zeocin

™

is light sensitive. Store drug, plates, and medium containing drug in

the dark.

• Wear gloves, a laboratory coat, and safety glasses or goggles when handling

solutions containing Zeocin

™

• Zeocin

™

is toxic. Do not ingest or inhale solutions containing the drug.

• Store tissue culture medium containing Zeocin

™

at 4°C in the dark. Medium

containing Zeocin

™

is stable for 1-2 months.

Map and Features of pPICZ A, B, and C

Map of pPICZ

A, B, and C

The figure below summarizes the features of the pPICZ A, B, and C vectors. The

complete sequences for pPICZ A, B, and C are available for downloading from

our web site at or from

Technical Support (page 32). See

the next page for a description of the features of the vector.

c-myc epitope

6xHis

Stop

BamH I

pPICZ A,B,C

3.3 kb

Comments for pPICZ A:

3329 nucleotides

Bgl II

5´ AOX1 promoter region: bases 1-941

5´ end of AOX1 mRNA: base 824

5´ AOX1 priming site: bases 855-875

Multiple cloning site: bases 932-1011

c-myc epitope tag: bases 1012-1044

Polyhistidine tag: bases 1057-1077

3´ AOX priming site: bases 1159-1179

3´ end of mRNA: base 1250

AOX1 transcription termination region: bases 1078-1418

Fragment containing TEF1 promoter: bases 1419-1830

EM7 promoter: bases 1831-1898

Sh ble ORF: bases 1899-2273

CYC1 transcription termination region: bases 2274-2591

pUC origin: bases 2602-3275 (complementary strand)

*The restriction site

between Not I and the

myc epitope is different

in each version of pPICZ:

Apa I in pPICZ A

Xba I in pPICZ B

SnaB I in pPICZ C

Continued on next page

Map and Features of pPICZ A, B, and C,

Continued

Features of pPICZ

A, B, and C

pPICZ A (3329 bp), pPICZ B (3328 bp), and pPICZ C (3329 bp) contain the

following elements. All features have been functionally tested.

Feature Benefit

5´ AOX1 promoter A 942 bp fragment containing the AOX1 promoter that

allows methanol-inducible, high-level expression of

the gene of interest in Pichia.

Targets plasmid integration to the AOX1 locus.

Multiple cloning site

c-myc epitope

(Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu)

Allows insertion of your gene into the expression

vector.

Permits detection of your recombinant fusion protein

with the Anti-myc Antibody or Anti-myc-HRP

Antibody (see page vi for ordering information)

(Evans et al., 1985).

Permits purification of your recombinant fusion

protein on metal-chelating resin such as ProBond

™

In addition, the C-terminal polyhistidine tag is the

epitope for the Anti-His(C-term) Antibody (page vi)

(Lindner et al., 1997) and the Anti-His(C-term)-HRP

Antibody (page vi).

AOX1 transcription termination (TT) region Native transcription termination and polyadenylation

signal from AOX1 gene (~260 bp) that permits efficient

3´ mRNA processing, including polyadenylation, for

increased mRNA stability.

C-terminal polyhistidine (6xHis) tag

TEF1 promoter Transcription elongation factor 1 gene promoter from

cerevisiae that drives expression of the

(GenBank accession numbers D12478, D01130)

Saccharomyces

™

Zeocin

resistance gene in Pichia.

EM7 promoter

Synthetic prokaryotic promoter that drives constitutive

expression of the Zeocin

™

resistance gene in E. coli.

Zeocin

™

resistance gene (Sh ble)

CYC1 transcription termination region

(GenBank accession number M34014)

pUC origin

Allows selection of transformants in E. coli and Pichia.

3´ end of the Saccharomyces cerevisiae CYC1 gene that

allows efficient 3´ mRNA processing of the Zeocin

™

resistance gene for increased stability.

Allows replication and maintenance of the plasmid in

E. coli.

Lithium Chloride Transformation Method

Introduction

Preparation of

Solutions

Preparation of

Cells

Transformation

This is a modified version of the procedure described for S. cerevisiae (Gietz &

Schiestl, 1996), and is provided as an alternative to transformation by

electroporation. Transformation efficiency is between 10

to 10

cfu/

g linearized

DNA.

Lithium acetate does not work with Pichia pastoris. Use only lithium chloride.

1 M LiCl in distilled, deionized water. Filter-sterilize. Dilute as needed with sterile

water.

50% polyethylene glycol (PEG-3350) in distilled, deionized water. Filter-sterilize.

Store in a tightly capped bottle.

2 mg/ml denatured, sheared salmon sperm DNA in TE (10 mM Tris-HCl, pH 8.0,

1.0 mM EDTA). Store at –20°C.

1. Grow a 50 ml culture of Pichia pastoris in YPD at 30°C with shaking to an OD

600

0.8 to 1.0 (approximately 10

cells/ml).

2. Harvest the cells, wash with 25 ml of sterile water, and centrifuge at 1500 × g for

10 minutes at room temperature.

3. Resuspend the cell pellet in 1 ml of 100 mM LiCl and transfer the suspension to a

1.5 ml microcentrifuge tube.

4. Pellet the cells at maximum speed for 15 seconds and remove the LiCl with a

pipet.

5. Resuspend the cells in 400

l of 100 mM LiCl.

6. Dispense 50

l of the cell suspension into a 1.5 ml microcentrifuge tube for each

transformation and use immediately. Do not store on ice or freeze

at –20°C.

1. Boil a 1 ml sample of single-stranded DNA for 5 minutes, then quickly chill on

ice. Keep on ice. Note: It is not necessary nor desirable to boil the carrier DNA

prior to each use. Store a small aliquot at –20°C and boil every

3–4 times the DNA is thawed.

2. Centrifuge the cells from Step 6, above, and remove the LiCl with a pipet.

3. For each transformation , add the following reagents in the order given to the

cells. PEG shields the cells from the detrimental effects of the high LiCl

concentration.

i. 240

l 50% PEG

ii. 36

l 1 M LiCl

iii. 25

l 2 mg/ml single-stranded DNA

iv. Plasmid DNA (5-10

g) in 50

l sterile water

4. Vortex each tube vigorously until the cell pellet is completely mixed (~1 minute).

5. Incubate the tube at 30°C for 30 minutes without shaking.

6. Heat shock in a water bath at 42°C for 20–25 minutes.

7. Centrifuge the cells at 6000 to 8000 rpm to pellet.

8. Resuspend the pellet in 1 ml of YPD and incubate at 30°C with shaking.

9. After 1 hour and 4 hours, plate 25–100

l on YPD plates containing the

appropriate concentration of Zeocin

™

. Incubate the plates for 2–3 days at 30°C.

Construction of In Vitro Multimers

Experimental

Outline

Alternative

Procedure

At this point you should have your gene cloned into the multiple cloning site of

either pPICZ A, B, or C. To generate multiple copies of your expression cassette:

Stage Description

1 Digest pPICZ containing your gene of interest with Bgl II and BamH I

to release the expression cassette (P

AOX1

plus your gene).

2 To clone multiple copies of the expression cassette, linearize pPICZ

containing your gene of interest using BamH I. Note that the

BamH I-linearized vector already contains one copy of your

expression cassette.

3 Treat the Bgl II-BamH I expression cassette with ligase in vitro. Note

that Bgl II and BamH I share 4 bases in common between their

recognition sites (GATC).

4 Generate head-to-tail, head-to-head, and tail-to-tail multimers

(Head-to-tail ligation, which is the correct orientation for expression,

will destroy both the BamH I and Bgl II sites).

5 Treat the ligation mix with BamH I and Bgl II to eliminate head-to-

head and tail-to-tail multimers.

6 Ligate into BamH I-linearized recombinant pPICZ.

7 Transform into E. coli and analyze recombinant plasmids for copy

number by digesting with Bgl II and BamH I.

You may wish to build each desired multimer in increments by ligating each

additional expression cassette one (or two) at a time into pPICZ A, B, or C. For

example:

Stage Description

1 Digest pPICZ containing one copy of your gene with BamH I.

2 Ligate a single copy of the Bgl II-BamH I expression cassette into

BamH I-digested vector.

3 Transform E. coli and analyze the transformants for the vector with

2 copies of your insert.

4 Isolate and digest this vector (with 2 copies of your gene) with

BamH I and Bgl II to release a cassette with 2 copies of your gene

(optional).

5 Digest the vector with 2 copies of your gene with BamH I and ligate

1 or 2 copies (see Step 4) of the expression cassette into the vector.

6 Transform E. coli and analyze the transformants for the vector with

3 or 4 copies of your insert.

7 Repeat until the desired multimer is reached.

Continued on next page

Construction of In Vitro Multimers,

Continued

Materials Needed

Controls

You will the following items:

• Electrocompetent or chemically competent E. coli (must be recA, endA) for

transformation (page v). You will need 3–4 tubes of competent cells per

experiment.

• BamH I and Bgl II restriction enzymes and appropriate buffers

• Low-melt agarose gel

• PureLink

™

Quick Gel Extraction Kit or S.N.A.P.

™

Gel Purification Kit (page

v) or glass milk

• Sterile water

• CIAP (calf intestinal alkaline phosphatase, 1 unit/μl, page v)

• 10X CIAP Buffer (supplied with CIAP, page v)

• Phenol/chloroform

• 3M sodium acetate

• 100% ethanol

• 80% ethanol

• T4 Ligase (2.5 units/μl, page v)

• 10X Ligation Buffer (with ATP)

• Low Salt LB plates containing 25

g/ml Zeocin

™

(page 17)

• 150 mm plates for plating transformants

• 16°C, 37°C, and 65°C water baths or temperature blocks

In order to evaluate your transformants and expression data later on, we

recommend transforming Pichia with pPICZ (the parent vector) and pPICZ

containing one copy of your gene of interest. This will allow you to compare

expression levels to see if multiple copies significantly increase the amount of

protein produced. Also, if you elect to determine how many copies of your gene

are in a recombinant by dot or Southern blot, the strain with the parent vector

will control for background hybridization and the strain with the single copy

gene will provide a signal to normalize your data.

Continued on next page

Construction of In Vitro Multimers,

Continued

Important

Digestion of

Recombinant

pPICZ

Production of

Expression

Cassettes for

Multimerization

Once you have created a pPICZ plasmid containing multimers, note that this

plasmid cannot be linearized because any enzyme that cuts in the 5´ AOX1

region will cut in all of the 5´ AOX1 regions present in the multimer. You can

transform with uncut plasmid, but you will need to use 50–100

g of DNA to

compensate for the 10 to 100-fold drop in transformation efficiency. However,

with selection on Zeocin

™

, any transformants you obtain will probably contain

your construct. For best results:

• Use electroporation to transform your cells.

• Use at least 50

g plasmid DNA for each transformation.

• Plate out all of the transformation mix on several YPDS plates containing the

appropriate concentration of Zeocin

™

. You will need to use the optional

outgrowth procedure on page 10.

Set up two separate digests of recombinant pPICZ containing one copy of your

gene:

1. Double digest 1-2

g of recombinant pPICZ in 20

l with 10 units each of

Bgl II and BamH I. Proceed to Production of Expression Cassettes for

Multimerization, Step 1.

2. Digest 2

g of recombinant pPICZ in 20

l with 10 units of BamH I only.

Proceed to Dephosphorylation of Vector, Step 1.

The S.N.A.P.

™

Gel Purification Kit available from Invitrogen (page v) allows you

to rapidly purify DNA fragments from regular agarose gels. Alternatively, you

may use glass milk. To use the S.N.A.P.

™

Gel Purification Kit, follow the steps

below:

1. Electrophorese your BamH I-Bgl II digest from Step1, above, on a 1 to 5%

regular TAE agarose gel. Note: Do not use TBE to prepare agarose gels.

Borate interferes with the sodium iodide step, below.

2. Cut out the gel slice containing the PCR product and melt it at 65°C in

2 volumes of the 6 M sodium iodide solution.

3. Add 1.5 volumes Binding Buffer.

4. Load solution (no more than 1 ml at a time) from Step 3 onto a PureLink

™

S.N.A.P.

™

spin column. Centrifuge 1 minute at 3000 × g in a microcentrifuge

and discard the supernatant.

5. If you have solution remaining from Step 3, repeat Step 4.

6. Add 900

l of the Final Wash Buffer.

7. Centrifuge 1 minute at full speed in a microcentrifuge and discard the

flow-through.

8. Repeat Step 7.

9. Elute the purified DNA in 15

l of sterile water. Store on ice if proceeding

immediately to Ligation of Expression Cassette, next page. Store at –20ºC

for long-term storage.

Continued on next page

Construction of In Vitro Multimers,

Continued

Dephosphorylation

Dephosphorylation of the BamH I-digested vector is necessary to prevent self-

ligation.

of Vector

1. Take your BamH I digest from Digestion of Recombinant pPICZ, Step 2 and

phenol extract, then ethanol precipitate the DNA. Resuspend in 17

l of

sterile water.

2. Set up a 20

l dephosphorylation reaction in a microcentrifuge tube as

follows:

• 17 μl BamH I digested recombinant pPICZ (page 24)

• 2 μl 10X CIAP Buffer

• 1 μl CIAP (1 Unit/μl)

3. Incubate at 37°C for 15 minutes.

4. Add 30

l of sterile water to the reaction for a final volume of 50

5. Add 50

l of phenol/chloroform and extract your DNA solution.

6. Precipitate the DNA by adding 5

l of 3 M sodium acetate and 110

l of 100%

ethanol. Incubate on ice for 30 minutes.

7. Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4°C.

Carefully decant the supernatant.

8. Wash the nucleic acid pellet with 80% ethanol, centrifuge 2 minutes, and

remove the ethanol.

9. Centrifuge again for 1 minute, remove residual ethanol, and air dry

the pellet.

10. Resuspend pellet in 8

l sterile water. Save on ice if you plan to ligate your

insert immediately (see Ligation and Digestion of Expression Cassette) or

store at –20°C.

Continued on next page

Construction of In Vitro Multimers,

Continued

Ligation and

Digestion of

Expression

Cassette

Ligation of the expression cassette will generate head-to-tail, head-to-head, and

tail-to-tail multimers. Creation of head-to-tail multimers will be in the correct

orientation for expression and will destroy both the BamH I and Bgl II sites

between the expression cassettes. Digestion of the multimers with BamH I and

Bgl II will eliminate those multimers with tail-to-tail and head-to-head orientation.

After digestion with these two restriction enzymes, you will have a mixture of

multimers containing 1, 2, 3, etc. copies of your gene that can be ligated into

BamH I-linearized, recombinant pPICZ.

1. Set up a 20

l ligation reactions as follows:

• 15 μl Bgl II-BamH I digested expression cassette

• 2 μl sterile water

• 2 μl 10X Ligation Buffer (with ATP)

• 1 μl T4 DNA Ligase (2.5 units/μl)

2. Incubate at 16°C for 2.5 hours.

3. Heat inactivate the ligase by incubating at 65°C for 20 minutes.

4. Add the following reagents for restriction enzyme digestion (cut-back).

Note: BamH I and Bgl II may be used with the same reaction buffer:

• 23 μl sterile water

• 5 μl 10X restriction enzyme buffer

• 1 μl Bgl II (10 units/μl)

• 1 μl BamH I (10 units/μl)

5. Incubate the reaction at 37°C for 2 hours.

6. Add 50 μl phenol/chloroform and extract the restriction enzyme digestion to

remove the enzymes. Transfer the aqueous solution to a new

microcentrifuge tube.

7. Add 5

l of 3 M sodium acetate and 110

l of 100% ethanol to ethanol

precipitate the DNA.

8. Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4°C.

Carefully decant the supernatant.

9. Wash the nucleic acid pellet with 80% ethanol, centrifuge 2 minutes, and

remove the ethanol. Centrifuge again for 1 minute, remove residual ethanol,

and air dry the pellet.

10. Resuspend pellet in 4 μl sterile water. Save on ice if you plan to ligate your

insert immediately or you can store at –20°C. Proceed to Ligation of

Multimers into Linearized Vector.

You may wish to combine the ligation reaction with the restriction enzyme

digestion to enrich for head-to-tail multimers. Use the reaction buffer for the

restriction enzymes and add 1 mM ATP to the reaction in order to ensure ligase

activity. Perform the reaction at 37°C. T4 ligase will retain most of its activity in the

restriction buffer. As head-to-head and tail-to-tail multimers form, they will be

digested, increasing the likelihood of obtaining head-to-tail multimers over time.

Continued on next page

Construction of In Vitro Multimers,

Continued

Ligation of

Multimers into

Linearized Vector

Transformation

into E. coli

You are now ready to ligate the mixture of multimers generated in Step 10, above,

into dephosphorylated, linearized vector.

1. Set up the following ligation reactions:

Dephosphorylated vector (page 27, Step 10) 4

Expression cassette multimers (Step 10, above) 4

10X Ligation Buffer 1

T4 DNA Ligase (2.5 units/μl) 1

Total volume 10

For the vector only control:

ephosphorylated vector 4

Sterile water 4 μl

10X Ligation Buffer 1

T4 DNA Ligase (2.5 units/μl) 1

Total volume 10

2. Incubate overnight at 16°C.

3. You can store the ligation reactions at –20°C until ready to use, or transform

1–10

l of each ligation mix into competent E. coli. Note that the amount of the

ligation mixture you transform depends on whether you use electrocompetent

or chemically competent cells. You may have to decrease the amount you to

transform into electrocompetent cells to prevent arcing.

Remember to include the "vector only" and "cells only" controls to evaluate your

experiment. The "vector only" will indicate whether your vector was

dephosphorylated. Since the CIAP reaction is not 100% and because you often

get degradation of the ends, there might be a few colonies on this plate. The

"cells only" plate should have no colonies at all.

1. Transform competent E. coli by your method of choice.

2. After adding medium to the transformed cells and allowing them to

recover, plate 10

l and 100

l of each transformation mix onto Low Salt LB

plates containing 25

g/ml Zeocin

™

(page 17). Save the remainder of your

transformation mix at 4°C.

3. Incubate overnight at 37°C. If you do not get transformants or very few

transformants, plate out the remainder of the transformation mix onto Low

Salt LB-Zeocin

™

plates.

Continued on next page

Construction of In Vitro Multimers,

Continued

Analysis of

Transformants

To analyze your transformants:

1. Pick 20 transformants and inoculate each colony into 2 ml Low Salt LB

containing 25

g/ml Zeocin

™

(page 17). Grow overnight at 37°C.

2. Isolate plasmid DNA and digest with Bgl II and BamH I to release any

multimers from pPICZ.

Note: Be sure to include Bgl II-BamH I digested pPICZ as a control. It is possible to

get vector rearrangements and deletions with large recombinant vectors in E. coli.

Including Bgl II-BamH I digested pPICZ will allow you to detect these

rearrangements-deletions in the vector backbone.

3. Analyze your digests on a 1% agarose gel. You should see bands

corresponding to 1 copy, 2 copies, 3 copies, etc. of your expression cassette

along with the vector backbone.

Note: The number of copies you obtain may depend on how well a large vector is

tolerated by the host strain.

4. Once you have identified plasmids with multiple copies of your expression

cassette, be sure to purify by streaking for single colonies and confirming

your construct.

5. Prepare frozen glycerol stocks of E. coli containing each of your multimeric

constructs.

6. Prepare at least 100

g of each plasmid for transformation into Pichia. You

need more DNA because you will be transforming with uncut plasmid

DNA. Transformation efficiency is about 1 to 2 orders of magnitude less for

uncut versus linearized DNA.

7. Proceed to Pichia Transformation, page 9. Use the outgrowth protocol on

page 10 to isolate transformants.

Continued on next page

Construction of In Vitro Multimers,

Continued

Troubleshooting

The table below will help you optimize formation and isolation of multimers in

Pichia.

Solution

Use fresh CIAP.

Problem Cause

No multimers or low number

CIAP defective

of multimers in your vector

after transformation into E. coli

Add more CIAP. Add 1 unit of CIAP

and incubate 15 more minutes at

37°C. This is somewhat risky as CIAP

can degrade the ends of your DNA.

Add more BamH I-Bgl II expression

cassette to your ligation.

Decrease the number of cassettes in

the vector.

Try ligating each expression cassette

stepwise (see page 28).

Decrease the number of cassettes in

the vector.

Transform using more DNA and/or

do multiple transformations with

more DNA and cells.

Not enough insert DNA to ligate

Construct is unstable in E. coli

Multimers are too long to ligate

efficiently

Recombinant vector rearranges Construct is unstable in E. coli

and deletions are detected

No Zeocin

™

-resistant Pichia

transformants

Integration efficiency is low

For More

Information

There are a number references in the literature you can consult in order to optimize

synthesis of in vitro multimers. A partial list is provided below:

Cohen, B. and Carmichael, G. G. (1986) A Method for Constructing Multiple Tandem

Repeats of Specific DNA Fragments. DNA 5: 339-343.

Eisenberg, S., Francesconi, S. C., Civalier, C. and Walker, S. S. (1990) Purification of

DNA-Binding Proteins by Site-specific DNA Affinity Chromatography. Methods

Enzymol. 182: 521-529.

Graham, G. J. and Maio, J. J. (1992) A Rapid and Reliable Method to Create Tandem

Arrays of Short DNA Sequences. BioTechniques 13: 780-789.

Rudert, W. A. and Trucco, M. (1990) DNA Polymers of Protein Binding Sequences

Generated by Polymerase Chain Reaction. Nucleic Acids Res. 18: 6460.

Simpson, R. T., Thoma, F. and Brubaker, J. M. (1985) Chromatin Reconstituted from

Tandemly-repeated Cloned DNA Fragments and Core Histones: A Model System for

the Study of Higher-order Structure. Cell 42: 799-808.

Takeshita, S., Tezuka, K.- i., Takahashi, M., Honkawa, H., Matsuo, A., Matsuishi, T.

and Hashimoto-Gotoh, T. (1988) Tandem Gene Amplification in vitro for Rapid and

Efficient Expression in Animal Cells. Gene 71: 9-18.

Taylor, W. H. and Hagerman, P. J. (1987) A General Method for Cloning DNA

Fragments in Multiple Copies. Gene 53: 139-144.

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Introduction

Limited Use Label

The Pichia Expression System is covered under the licenses detailed below.

This product is licensed under U.S. Patent Nos. 5,284,933 and 5,310,663 and foreign equivalents from

License No. 22

Vectors and

Clones Encoding

Histidine Hexamer

Limited Use Label

License No. 74

Pichia Pastoris

Expression

System

Hoffmann-LaRoche, Inc., Nutley, NJ and/or Hoffmann-LaRoche Ltd., Basel, Switzerland and is

provided only for use in research. Information about licenses for commercial use is available from

QIAGEN GmbH, Max-Volmer-Str. 4, D-40724 Hilden, Germany.

The Pichia Expression System is based on the yeast Pichia pastoris. Pichia pastoris was developed into

an expression system by scientists at Salk Institute Biotechnology/ Industry Associates (SIBIA) and

Phillips Petroleum for high-level expression of recombinant proteins. All patents for Pichia pastoris

and licenses for its use as an expression system are owned by Research Corporation Technologies

(RCT), Inc., Tucson, Arizona. Life Technologies has an exclusive license to sell Pichia expression kits

and vectors to scientists for research purposes only, under the terms described below. Use of

Pichia

pastoris by commercial entities for any commercial purpose requires the user to obtain a commercial

license as detailed below. Before using any Pichia expression product, please read the following

license agreement. If you do not agree to be bound by its terms, contact Life Technologies within 10

days for authorization to return the unused Pichia expression products and to receive a full refund. If

you do agree to the terms of this license agreement, please complete the User Registration Card and

return it to Life Technologies before using the product.

Life Technologies Corporation ("Life Technologies") grants you a non-exclusive license to use the

enclosed Pichia expression vectors ("Expression Vector") for academic research or for evaluation

purposes only. The Expression Vectors are being transferred to you in furtherance of, and reliance on,

such license. You may not use the Expression Vectors for any commercial purpose without a license

for such purpose from Research Corporation Technologies, Inc., Tucson, Arizona.

Commercial purposes include: any use of Expression Products or Expression Vectors in a Commercial

Product; any use of Expression Products or Expression Vectors in the manufacture of a Commercial

Product; any sale of Expression Products; any use of Expression Products or the Expression Kit to

facilitate or advance research or development directed to a Commercial Product; and any use of

Expression Products or the Expression Kit to facilitate or advance any research or development

program the results of which will be directly applied to the development or manufacture of a

Commercial Product. "Expression Products" means products expressed with the Expression Kit, or

with the use of any Pichia expression vectors (including the Expression Vector) or host strains.

"Commercial Product" means any product intended for sale or commercial use.

Commercial entities may conduct their evaluation for one year at which time this license

automatically terminates. Commercial entities will be contacted by Research Corporation

Technologies during the evaluation period regarding their desire for a commercial license.

Access to the Expression Kit and Vector must be limited solely to those officers, employees and

students of your institution who need access to perform the above-described research or evaluation.

You must inform each such officer, employee and student of the provisions of this license agreement

and require them to agree, in writing, to be bound by the provisions of this license agreement. You

may not distribute any Expression Vector or host strain contained herein or in the Expression Kit to

others, even those within your own institution. You may only transfer modified, altered, or original

material from the Expression Kit or Vector to a third party following written notification of, and

written approval from, Life Technologies so that the recipient can be licensed. You may not assign,

sub-license, rent, lease or otherwise transfer this license agreement or any of the rights or obligation

there under, except as expressly permitted by Life Technologies and RCT.

This license agreement is effective until terminated. You may terminate it at any time by destroying

all Pichia Expression products in your control. It will also terminate automatically if you fail to

comply with the terms and conditions of the license agreement. You shall, upon termination of the

license agreement, destroy all Pichia Expression products in your control, and so notify Life

Technologies in writing.

You may contact Research Corporation Technologies at the following address: Bennett Cohen, Ph.D.,

Research Corporation Technologies, 101 North Wilmot Road, Suite 600, Tucson, Arizona 85711-3335.

Tel: 520-748-4443, Fax: 520-748-0025.

References

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current

Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York

Baron, M., Reynes, J. P., Stassi, D., and Tiraby, G. (1992) A Selectable Bifunctional b-Galactosidase: Phleomycin-

resistance Fusion Protein as a Potential Marker for Eukaryotic Cells. Gene 114, 239-243

Calmels, T., Parriche, M., Burand, H., and Tiraby, G. (1991) High Efficiency Transformation of Tolypocladium geodes

Conidiospores to Phleomycin Resistance. Curr. Genet. 20, 309-314

Drocourt, D., Calmels, T. P. G., Reynes, J. P., Baron, M., and Tiraby, G. (1990) Cassettes of the Streptoalloteichus

hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance. Nucleic

Acids Res. 18, 4009

Ellis, S. B., Brust, P. F., Koutz, P. J., Waters, A. F., Harpold, M. M., and Gingeras, T. R. (1985) Isolation of Alcohol

Oxidase and Two other Methanol Regulatable Genes from the Yeast, Pichia pastoris. Mol. Cell. Biol. 5, 1111-

1121

Evans, G. I., Lewis, G. K., Ramsay, G., and Bishop, V. M. (1985) Isolation of Monoclonal Antibodies Specific for c-myc

Proto-oncogene Product. Mol. Cell. Biol. 5, 3610-3616

Gietz, R. D., and Schiestl, R. H. (1996) in Methods in Molecular and Cellular Biology, in press

Henikoff, S., and Cohen, E. H. (1984) Sequences Responsible for Transcription Termination on a Gene Segment in

Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 1515-1520

Higgins, D. R., and Cregg, J. M. (eds) (1998) Pichia Protocols Vol. 103. Methods in Molecular Biology. Edited by

Walker, J. M., Humana Press, Totowa, NJ

Irniger, S., Egli, C. M., and Braus, G. H. (1991) Different Classes of Polyadenylation Sites in the Yeast Saccharomyces

cerevisiae. Mol. Cell. Bio. 11, 3060-3069

Koutz, P. J., Davis, G. R., Stillman, C., Barringer, K., Cregg, J. M., and Thill, G. (1989) Structural Comparison of the

Pichia pastoris Alcohol Oxidase Genes. Yeast 5, 167-177

Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R., and

Pluckthun, A. (1997) Specific Detection of His-tagged Proteins With Recombinant Anti-His Tag scFv-

Phosphatase or scFv-Phage Fusions. BioTechniques 22, 140-149

Mulsant, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1988) Phleomycin Resistance as a Dominant Selectable Marker in

CHO Cells. Somat. Cell Mol. Genet. 14, 243-252

Perez, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1989) Phleomycin Resistance as a Dominant Selectable Marker for

Plant Cell Transformation. Plant Mol. Biol. 13, 365-373

Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992) Foreign Gene Expression in Yeast: A Review. Yeast

8, 423-488

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual

, Second Ed., Cold Spring

Harbor Laboratory Press, Plainview, New York

Scorer, C. A., Buckholz, R. G., Clare, J. J., and Romanos, M. A. (1993) The Intracellular Production and Secretion of

HIV-1 Envelope Protein in the Methylotrophic Yeast Pichia pastoris. Gene 136, 111-119

Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C., and Gingeras, T. R. (1987a) Expression of the lacZ Gene from Two

Methanol Regulated Promoters in Pichia pastoris. Nucleic Acids Res. 15, 3859-3876

Zaret, K. S., and Sherman, F. (1984) Mutationally Altered 3´ Ends of Yeast CYC1 mRNA Affect Transcript Stability

and Translational Efficiency. J. Mol. Biol. 177, 107-136

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owners.

Notes