43
Analysis by SDS-Polyacrylamide Gel Electrophoresis,
continued

Preparing
Samples,
continued Preparing supernatant (Secreted Expression only):
1. Thaw supernatants and place on ice.
2. Mix 50 µl of the supernatant with 50 µl of SDS-PAGE Gel Loading buffer.
3. Boil 10 minutes, then load 10…30 µl onto the gel. You may store the
remaining sample at …20°C for Western blots, if necessary. You may store the
supernatants at …80°C for further analysis.
4. If you do not see any protein by Coomassie or by Western blot, then
concentrate the supernatant 5…10 fold and analyze samples again by Western
blot. Centricon and Centriprep filters (Amicon) are very useful for this
purpose.

Concentrating
Protein You may perform Lowry, BCA (Pierce) or Bradford protein assays to quantify
the amounts of protein in the cell lysates and medium supernatants. In general,
Pichia cell lysates contain 5…10 µg/µl protein. Pichia medium supernatants will
vary in protein concentration primarily due to the amount of your secreted
protein. Pichia secretes very few native proteins. If the protein concentration of
the medium is > 50 µg/ml, 10 µl of medium will give a faint band on a
Coomassie-stained SDS-PAGE gel.

Controls Include the following samples as controls on your SDS-PAGE:
€ Molecular weight standards appropriate for your desired protein
€ A sample of your protein as a standard (if available)
€ A sample of X-33, GS115, or KM71H with the parent plasmid transformed
into it. This shows the background of native Pichia proteins that are present
intracellularly. Inclusion of this sample will help you differentiate your
protein from background if you express it intracellularly.
€ Analyze the GS115/pPICZ/lacZ and Albumin controls also as they should
indicate any problems with the media or expression conditions.















In addition to Coomassie-stained SDS-PAGE, we strongly recommend
performing a Western blot or another more sensitive assay to detect your
protein. Visualization of the expressed protein will depend on several factors
including its expression level, its solubility, its molecular weight, and whether it
will be masked by an abundant cellular protein of the same size. Western blot
analysis, enzymatic activities, or a defined purification profile, if available, may
help to identify the expressed protein among the native Pichia cellular proteins.
Continued on next page

44
Analysis by SDS-Polyacrylamide Gel Electrophoresis,
continued

Western Blot
Analysis To detect expression of your recombinant fusion protein by Western blot analysis,
you may use Anti-myc or Anti-His(C-term) antibodies available from Invitrogen
(see page viii) or an antibody to your protein of interest. In addition, the
Positope
’
Control Protein (Cat. no. R900-50) is available for use as a positive
control for detection of fusion proteins containing a c-myc epitope or a
polyhistidine tag. The ready-to-use WesternBreeze
’
Chromogenic Kits and
WesternBreeze
’
Chemiluminescent Kits are available from Invitrogen to facilitate
detection of antibodies by colorimetric or chemiluminescent methods. For more
information, refer to our website (www.invitrogen.com) or call Technical Support
(see page 78).

Analyzing Protein
Expression Inspection of your Coomassie-stained SDS-PAGE should reveal the induction
over time of your protein co-migrating with your standard. If you are satisfied
with the level of expression, try a test purification (page 50) or scale-up expression
(page 47).
If there is no recombinant protein visible, then perform either a Western blot or a
funct
ional assay if available.
If you detect low expression of your recombinant protein, see Optimizing Pichia
Protein Expression, page 45, for guidelines to optimize expression.
Test your expression conditions with the one of the two control strains included
in t
he kit (GS115/pPICZ/lacZ or GS115/Albumin).
If there is no indication of expression at all, use PCR to analyze your
recombinants for the correctly sized PCR product (page 69). If you find that you
hav
e recombinants, perform a Northern analysis to see if and how much full-
length mRNA is induced. See page 75 for an RNA isolation protocol.

45
Optimizing Pichia Protein Expression

Introduction Based on available data, there is approximately a 50 to 75% chance of expressing
your protein of interest in Pichia pastoris at reasonable levels. The biggest hurdle
seems to be generating initial success--i.e., expression of your protein at any level.
While there are relatively few examples of expression of >10 grams/liter, there
are many examples of expression in the >1 gram/liter range, making the Pichia
pastoris expression system one of the most productive eukaryotic expression
systems available. Likewise, there are several examples of proteins that have been
successfully expressed in Pichia pastoris that were completely unsuccessful in
baculovirus or Saccharomyces cerevisiae, suggesting that the Pichia pastoris system is
an important alternative to have available. If you obtain no or low protein
expression in your initial expression experiment, use the following guidelines to
optimize expression.

Proteolysis or
Degradation € Do a time course study of expression. Check to see if there is a time point that
yields a larger percentage of full-length protein.
€ If secreting your protein, check to see if your protein is susceptible to neutral
pH proteases by expressing in unbuffered medium (MMH). In addition, try
1% Casamino acids with buffered medium to inhibit extracellular proteases.
Low Secreted
Expression Levels € Check cell pellet to see if overall expression is low or if the protein did not
secrete. If it did not secrete, try a different signal sequence (e.g., a native or
D-factor signal sequence).
€ Concentrate your supernatant by ammonium sulfate precipitation or
ultrafiltration (see page 49).
€ For
Mut
+
, induce expression with a higher density culture.

Low Expression Levels € Look for multi-copy recombinants (i.e., jackpot clones) by dot blot (see
page 73). There are quite a few examples of increasing the expression levels
of a particular protein by increasing the gene dosage. See (Clare et al., 1991a;
Cla
re et al., 1991b; Romanos et al., 1991).
€ Check both Mut
+
and Mut
S
recombinants for increased expression. Some
proteins express better in one type of genetic background than another.
€ If secreting your protein, try intracellular expression. The protein may not be
processed correctly and fail to secrete. Be sure you check your cell pellets for
evidence of expression. If you are having problems with intracellular
expression, try secreting your protein. It probably will glycosylate, which
may be desirable or not. If glycosylation is undesirable, oligosaccharides can
be removed with Peptide:N-Glycosidase F (New England BioLabs).
€ Scale up to fermentation (page 49). Pichia is a yeast, and is particularly well
suited to growth in a
fermentor.
Continued on next page

46
Optimization of Pichia Protein Expression, continued

No Expression Be sure to try some of the easier things listed above as no expression can be the
same thing as very low expression. If none of these things improve protein
expression, use PCR to check for insertion of your gene into the Pichia genome
(page 68). If your gene is present, perform a Nort
hern blot analysis to check for
transcription of your gene. There is a protocol in the Appendix for RNA
isolation from Pichia (see page 75).
If you see premature transcriptional termination, check the AT content of your
gene. In Saccharomyces, there are a few consensus sequences which promote
prema
ture termination. One of these, TTTTTATA, resembles a sequence in HIV-1
gp120, ATTATTTTAT AAA, which prematurely terminatates mRNA when
expressed in Pichia . When this sequence was changed, longer transcripts were
found (Scorer et al., 1993).

Hyper-
glycosylation If your protein is hyperglycosylated:
€ Try intracellular expression as your protein will not go through the secretion
pathway and therefore, not be modified.
€ Try deglycosylating the protein with Peptide:N-Glycosidase F or other
enzymes (see page 52).

47
Scale-up of Expression

Guidelines for
Expression Once expression is optimized, you will want to scale-up your expression protocol
to produce more protein. This may be done by increasing the culture volume
using larger baffled flasks (below) or fermentation. Use the guidelines below to
scale-up your expression protocol. To purify your protein, see page 50.
Mut
+
Intracellular
or Secreted 1. Using a single colony, inoculate 25 ml of MGYH, BMGH, or BMGY in a
250 ml baffled flask. Grow at 28…30°C in a shaking incubator (250…300 rpm) until culture reaches an OD
600 = 2…6 (approximately 16…18 hours).
2. Use this 25 ml culture to inoculate 1 liter of MGYH, BMGH, or BMGY in a
3 or 4 liter baffled flask and grow at 28…30°C with vigorous shaking
(250…300 rpm) until the culture reaches log phase growth (OD
600 = 2…6).
3. Harvest the cells using sterile centrifuge bottles by centrifuging at
1500…3000 u g for 5 minutes at room temperature. To induce expression,
decant the supernatant and resuspend cell pellet to an OD
600 = 1.0 (2…6 liters)
in MMH, BMMH, or BMMY medium to start induction.
4. Aliquot the culture between several 3 or 4 liter baffled flask. Cover the flasks
with 2 layers of sterile gauze or cheesecloth and return to incubator. Continue to grow at 28…30°C with shaking.
5. Add 100% methanol to 0.5% every 24 hours until the optimal time of
induction is reached as determined from the time course study.
6. Harvest cells by centrifuging at 1,500…3,000 u g for 5 minutes at room
temperature.
For intracellular expression, decant the supernatant and store the cell pellets at …80°C until ready to process.
For secreted expression, save the supernatant, chill to 4°C, and concentrate it
down if desired (see page 49). Proceed directly to purification (page 50) or store
t
he supernatant at …80°C until ready to process further.
Continued on next page

48
Scale-up of Expression, continued

Mut
S
Intracellular
or Secreted 1. Using a single colony, inoculate 10 ml of MGYH, BMGH, or BMGY in a
100 ml baffled flask. Grow at 28…30°C in a shaking incubator (250…300 rpm)
until the culture reaches an OD
600 = 2…6 (approximately 16…18 hours).
2. Use this 10 ml culture to inoculate 1 liter of MGYH, BMGH, or BMGY in a
3 or 4 liter baffled flask and grow at 28…30°C with vigorous shaking
(250…300 rpm) until the culture reaches log phase growth (OD
600 = 2…6).
3. Harvest the cells by centrifuging at 1,500…3,000 u g for 5 minutes at room
temperature. To induce expression, decant the supernatant and resuspend cell
pellet in 1/5 to 1/10 of the original culture volume of MMH, BMMH, or
BMMY medium (approximately 100…200 ml).
4. Place the culture in a 1 liter baffled flask. Cover the flask with 2 layers of
sterile gauze or cheesecloth and return to incubator. Continue to grow at
28…30°C with shaking.
5. Add 100% methanol to 0.5% every 24 hours until the optimal time of
induction is reached.
6. Harvest cells by centrifuging at 1,500…3,000 u g for 5 minutes at room
temperature.
For intracellular expression, decant the supernatant and store the cell pellets at
…80°C until ready to process.
For secreted expression, save the supernatant, chill to 4°C, and concentrate it
down if desired (see next page). Proceed directly to purification (page 50) or store
t
he supernatant at …80°C until ready to process further.


To increase the amount of cells for Mut
S
recombinants, increase the number of
flasks, put 200…300 ml in a 3 liter flask, or try fermentation.
Continued on next page

49
Scale-up of Expression, continued

Concentration of
Proteins Proteins secreted into the media are usually > 50% homogeneous and will
require some additional purification (see page 50 or 52). It is optimal to
concentrate the protein if the expression level is not particularly high. There are
several general methods to concentrate proteins secreted from Pichia. These
general m
ethods include:
€ Ammonium sulfate precipitation
€ Dialysis
€ Centrifuge concentrator for small volumes (e.g., Centricon or Centriprep
devices available from Amicon)
€ Pressurized cell concentrators for large volumes (Amicon ultrafiltration
devices)
€ Lyophilization
A general guide to protein techniques is Protein Methods (Bollag and Edelstein,
1991).

Cell Lysis A general procedure for cell lysis using glass beads is provided on the next page.
There is also a cell lysis protocol in Current Protocols in Molecular Biology,
page 13.13.4. (Ausubel et al., 1994) and in Guide to Protein Purification (Deutscher,
1990). We also recommend lysis by French Press (follow the manufacturer's
suggestions for yeast).

Fermentation Basic guidelines are available for fermentation of Pichia from Invitrogen. We
recommend that only those with fermentation experience or those who have
access to people with experience attempt fermentation. Contact Technical
Support (see page 78) for more information.

50
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
’
.
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
that 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 (Cat. no. K850-01)
for purifying fusion proteins expressed from pPICZ or pPICZD. Note that
instructions for equilibration of and chromatography on ProBond
’
resin are
contained in the ProBond
’
Purification System.
If you are using a metal-chelating resin other than ProBond
’
, follow the
manufacturer's recommendations for 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 nature of 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 at 4°C.
Preparing Cell
Lysates Express your protein using a small-scale culture (10…20 ml for Mut
S
strains;
100…200 ml for Mut
+
) and the optimal conditions for expression (if determined).
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 Recipes , page 59.
1.
Wash cells once in BB by resuspending them and centrifuging 5…10 minutes
at 3,000 u 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 u 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

51
Purification, continued

Sample
Application
(Native
Conditions) 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 u 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.

Sample
Application
(Denaturing
Conditions) 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 some Pichia proteins may be retained on the ProBond
’

column using native purification conditions. Optimization of the purification or
using denaturing purification may remove these non-specific Pichia proteins (see
ProBond
’
Purification System manual).

Analysis of
Purification 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.

Scale-up 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 System)
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.

52
Protein Glycosylation

Analyzing
Glycoproteins When expressing and purifying a glycosylated protein in a heterologous
expression system, it is desirable to quickly determine whether the protein is
glycosylated properly. There are published protocols for carbohydrate analysis
of proteins to allow the molecular biologist to characterize glycosylated proteins
of interest (Ausubel et al., 1994), Unit 17. Further information about glycosylation
in eukaryotes is available in a review by Varki and Freeze (Varki and Freeze,
1994).
Enzymes for
Analyzing
Glycoproteins These are just a few of the enzymes available for carbohydrate analysis.
Abbreviations are as follows: Asn - Asparagine, Gal - Galactose,
GlcNAc - N-acetylglucosamine, GalNAc - N-acetylgalactosamine, and
NeuAc - N-acetylneuraminic acid.
Enzyme Type of
enzyme
Specificity
Endoglycosidase D Endo Cleaves various high mannose
glycans
Endoglycosidase F Endo Cleaves various high mannose
glycans
Endoglycosidase H Endo Cleaves various high mannose
glycans
E-galactosidase Exo Removes terminal galactosides from
Gal-E1,3-GlcNAc, Gal-E 1,4-GlcNAc
or Gal-E1,3 GalNAc.
Peptide:N-Glycosidase F Endo Glycoproteins between Asn and
GlcNAc (removes oligosaccharides)
Sialidases (Neuraminidases)
Vibrio cholerae
Clostridium perfringens
Arthobacter ureafaciens
Newcastle disease virus
Exo NeuAc-D2,6-Gal,
NeuAc-D2,6-GlcNAc
or NeuAc-D2,3-Gal

Commercial
Carbohydrate
Analysis There are a number of commercial vendors who will contract to analyze proteins
for glycosylation. A number of companies also supply kits and reagents for
researchers to do carbohydrate analysis in their own laboratories. A partial list is
provided below:
Company Type of Service Phone Number
Glyko Kits for Carbohydrate Analysis,
Reagents, Contract Services
1-800-334-5956
Oxford GlycoSystems Kits for Carbohydrate Analysis,
Reagents, Contract Services
1-800-722-2597
New England BioLabs Reagents 1-800-632-5227

54
Pichia Media Recipes

Introduction The expression of recombinant proteins in Pichia pastoris requires the preparation
of several different media. Recipes for these media are included in this section. In
addition, Yeast Nitrogen Base is available from Invitrogen (see below for
ordering information).
Item Amount Cat. no.
67 g pouch

Each pouch contains reagents to prepare 500 ml of a 10X YNB solution
Q300-07
Yeast Nitrogen Base
…with ammonium sulfate
…without amino acids
500 g Q300-09

Stock Solutions 10X YNB (13.4% Yeast Nitrogen Base with Ammonium Sulfate without amino
acids)
Dissolve 134 g of yeast nitrogen base (YNB) with ammonium sulfate and without
amino acids in 1000 ml of water and filter sterilize. Heat the solution to dissolve
YNB completely in water. Store at 4°C. Alternatively, use 34 g of YNB without
ammonium sulfate and amino acids and 100 g of ammonium sulfate. The shelf
life of this solution is approximately one year. If you are using the YNB pouch
included in the kit, follow the directions on the pouch.
Note: Pichia cells exhibit optimal growth with higher YNB concentrations; therefore, the
amount of YNB used in this kit is twice as concentrated as YNB formulations for
Saccharomyces.

500X B (0.02% Biotin)
Dissolve 20 mg biotin in 100 ml of water and filter sterilize. Store at 4°C.
The shelf life of this solution is approximately one year.
100X H (0.4% Histidine)
Dissolve 400 mg of L-histidine in 100 ml of water. Heat the solution, if necessary,
to no greater than 50°C in order to dissolve. Filter sterilize and store at 4°C.
The shelf life of this solution is approximately one year.
10X D (20% Dextrose)
Dissolve 200 g of D-glucose in 1000 ml of water. Autoclave for 15 minutes or
filter sterilize. The shelf life of this solution is approximately one year.
10X M (5% Methanol)
Mix 5 ml of methanol with 95 ml of water. Filter sterilize and store at 4°C.
The shelf life of this solution is approximately two months.
10X GY (10% Glycerol)
Mix 100 ml of glycerol with 900 ml of water. Sterilize either by filtering or
autoclaving. Store at room temperature. The shelf life of this solution is greater
than one year.
1 M potassium phosphate buffer, pH 6.0:
Combine 132 ml of 1 M K
2HPO4, 868 ml of 1 M KH2PO4 and confirm that the pH
= 6.0 ± 0.1 (if the pH needs to be adjusted, use phosphoric acid or KOH). Sterilize
by autoclaving and store at room temperature. The shelf life of this solution is
greater than one year.
Continued on next page

60
Proteins Expressed in Pichia

Table The table below provides a partial list of references documenting successful
expression of heterologous proteins in Pichia pastoris. Note that both Mut
+
and
Mut
S
phenotypes were used successfully as well as secreted and intracellular
expression.

Protein Expression
Levels
(grams/liter)
Where Expressed
How Expressed
Reference
Enzymes
Invertase 2.3 Secreted
Mut
+

(Tschopp et al. ,
1987b)
Bovine Lysozyme c2 0.55 Secreted
Mut
+

(Digan et al., 1989)
Streptokinase
(active)
0.08 Intracellular
*
(Hagenson et al.,
1989)
Alpha amylase 2.5 Secreted
Mut
S

(Paifer et al., 1994)
Pectate Lyase 0.004 Secreted
Mut
S

(Guo et al., 1995)
Spinach Phospho- ribulokinase
0.1 Intracellular
Mut
S

(Brandes et al.,
1996)
Antigens
Hepatitis B surface antigen
0.4 Intracellular
Mut
S

(Cregg et al., 1987)
Pertussis Antigen P69
3.0 Intracellular
Mut
S

(Romanos et al.,
1991)
Tetanus Toxin
Fragment C
12.0 Intracellular
Mut
+
/Mut
S

(Clare et al., 1991a)
HIV-1 gp120 1.25 Intracellular
Mut
+

(Scorer et al., 1993)
Tick Anticoagulant
protein
1.7 Secreted
Mut
S

(Laroche et al.,
1994) Bm86 Tick Gut Glycoprotein
1.5 Secreted
*
(Rodriguez et al.,
1994)
Continued on next page

61
Proteins Expressed in Pichia, continued

Proteins Expressed in Pichia, continued

Protein Expression
Levels
(grams/liter)
Where Expressed
How Expressed
Reference
Regulatory
Proteins

Tumor Necrosis
Factor (TNF)
10.0 Intracellular
Mut
S

(Sreekrishna et al.,
1989)
Mouse Epidermal Growth Factor (EGF)
0.45 Secreted
Mut
S

(Clare et al., 1991b)
Human Interferon
(IFN) D2b
0.4 Intracellular
Mut
S

(Garcia et al., 1995)
Membrane Proteins
Human CD38
(soluble portion)
0.05 Secreted
Mut
S

(Fryxell et al. , 1995)
Mouse Serotonin Receptor
0.001 Secreted
Mut
+

(Weiss et al., 1995)
Proteases and
Protease Inhibitors

Carboxypeptidase B 0.8 Secreted
Mut
+
/Mut
S

(Despreaux and Manning, 1993)
Enterokinase 0.021 Secreted
Mut
+

(Vozza et al., 1996)
Ghilanten 0.01 Secreted
Mut
+

(Brankamp et al.,
1995) Kunitz protease inhibitor
1.0 Secreted
*
(Wagner et al. ,
1992)
Human Proteinase Inhibitor 6
0.05 Intracellular
Mut
+

(Sun et al., 1995)
Antibodies
Rabbit Single Chain Antibody
>0.1 Secreted
Mut
S

(Ridder et al., 1995)
* Mut phenotype was not described in the paper.

63
Recombination and Integration in Pichia, continued

Multiple Gene
Insertion Events Multiple gene insertion events at a single locus in a cell do occur spontaneously
with a low, but detectable frequency--between 1 and 10% of all selected Zeo
R

transformants. Because of the low frequency of multiple gene insertion events,
you will need to screen hundreds to thousands of Zeocin
’
-resistant transformants
to locate these "jack-pot" clones. We recommend that you use electroporation to
generate Zeo
R
transformants for screening.
Multi-copy events can occur as gene insertions either at the AOX1 or the
aox1::ARG4 loci. This results in a Mut
+
phenotype in X-33 or GS115 and a Mut
S

phenotype in KM71H. Multiple gene insertion events can be detected by
quantitative dot blot analysis, Southern blot analysis, and differential
hybridization. See page 73 for a protocol to screen for m
ultiple inserts.

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ZeocinGene of InterestTT(
3' AOX1
Expression Cassette 1
2nd Insertion Event
Expression
Cassette 1
3rd Insertion Event, etc.
( (
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Expression Cassette 2
ZeocinGene of InterestTT
5´ PAOX1
5´ PAOX1
Zeo
c
i
n
G
e
n
e
o
f In
te
rest
T
T
5´ TTAOX1 or aox1::ARG4 3´
5´ TTAOX1 or aox1::ARG4 3´
5´ PAOX1

64
Lithium Chloride Transformation Method

Introduction This is a modified version of the procedure described for S. cerevisiae (Gietz and
Schiestl, 1996). This protocol is provided as an alternative to transformation by
electroporation. Transformation efficiency is between 10
2
to 10
3
cfu/µg linearized
DNA.

Preparation of
Solutions 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, fragmented salmon sperm DNA in TE (10 mM Tris-HCl,
pH 8.0, 1.0 mM EDTA). Store at …20°C.
Preparing Cells 1. Grow a 50 ml culture of Pichia pastoris in YPD at 30°C with shaking to an
OD
600 of 0.8 to 1.0 (approximately 10
8
cells/ml).
2. Harvest the cells and wash with 25 ml of sterile water and centrifuge at
1,500 u g for 10 minutes at room temperature.
3. Decant the water and resuspend the cells in 1 ml of 100 mM LiCl.
4. Transfer the cell suspension to a 1.5 ml microcentrifuge tube.
5. Pellet the cells at maximum speed for 15 seconds and remove the LiCl with
a pipet.
6. Resuspend the cells in 400 µl of 100 mM LiCl.
7. Dispense 50 µl 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.
Continued on next page

65
Lithium Chloride Transformation Method, continued

Transformation 1. Boil a 1 ml sample of single-stranded DNA for five minutes, then quickly
chill in ice water. 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 LiCl-cell solution from Step 7, above, and remove the LiCl
with a pipet.
3. For each transformation sample, add the following reagents in the order
given to the cells. PEG shields the cells from the detrimental effects of the
high concentration of LiCl.
240 µl 50% PEG
36 µl 1 M LiCl
25 µl 2 mg/ml single-stranded DNA
Plasmid DNA (5…10 µg) in 50 µl 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 tubes at 6,000 to 8,000 rpm and remove the transformation
solution with a pipet.
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 µl to 100 µl on YPD plates containing
100 µg/ml Zeocin
’
. Incubate the plates for 2…3 days at 30°C. Proceed to
Analysis of Pichia Transformants, page 28.

66
Zeocin
™


Description Zeocin
’
belongs to a family of structurally related bleomycin/phleomycin-type
antibiotics isolated from Streptomyces. Antibiotics in this family are broad
spectrum antibiotics that act as strong antibacterial and anti-tumor drugs. They
show strong toxicity against bacteria, fungi (including yeast), plants, and
mammalian cells. Zeocin
’
is not as toxic as bleomycin on fungi.

Chemical
Properties Zeocin
’
is a basic, water-soluble compound isolated from Streptomyces verticillus
as a copper-chelated glycopeptide. The presence of copper gives the solution its
blue color. The chemical formula for Zeocin
’
is C55H83N19O21S2Cu. It contains
several unique amino acids, sugars, and aliphatic amines. For general
information about the family of bleomycin antibiotics, See Berdy, 1980. The
general structure of Zeocin
’
is shown below.










 






























 










 


 
 



 


Mechanism of
Action The exact mechanism of action of Zeocin
’ is not known; however, it is thought to
be the same as bleomycin and phleomycin due to its similarity to these drugs
and its inhibition by the Sh ble resistance protein (see next section). The
copper/glycopeptide complex is selective and involves chelation of copper
(Cu
2+
) by the amino group of the D-carboxamide, single nitrogen atoms of both
the pyrimidine chromophore and the imidazole moiety, and the carbamoyl
group of mannose. The copper-chelated form is inactive. When the antibiotic
enters the cell, the copper cation is reduced from Cu
2+
to Cu
1+
and removed by
sulfhydryl compounds in the cell. Upon removal of the copper, Zeocin
’
is
activated to bind DNA and cleave it causing cell death (Berdy, 1980). High salt
concentrations and acidity or basicity inactivate Zeocin
’
; therefore, it is
necessary to reduce the salt in bacterial medium to 90 mM (5 g/liter) or less
and adjust the pH to 7.5 to make sure the drug remains active.
Continued on next page

67
Zeocin
™
, continued

Resistance to
Zeocin
™

A Zeocin
’ resistance protein has been isolated and characterized (Calmels et al.,
1991; Drocourt et al., 1990; Gatignol et al., 1988). This protein, the product of the
Sh ble gene (Streptoalloteichus hindustanus bleomycin gene), is a 13,665 Da protein
that binds Zeocin
’
in a stoichiometric manner. The binding of Zeocin
’
inhibits
its DNA strand cleavage activity. Expression of this protein in eukaryotic and
prokaryotic hosts confers resistance to Zeocin
’
. The nucleic acid and protein
sequence is given below:

 

 
 







3
3
!$3












69
PCR Analysis of Pichia Integrants, continued

Interpreting PCR If screening Mut
+
integrants, you should see two bands, one corresponding to
the size of your gene of interest, the other to the AOX1 gene (approximately
2.2 kb). In KM71H, the PCR product is 3.6 kb because of the ARG4 insert in
AOX1. Parent plasmids will produce the following sized PCR products. Add the
size of these products to the size of your insert to interpret your PCR results.
Vector PCR Product
pPICZ 325 bp (A), 323 bp (B), 324 bp (C)
pPICZD (using the 5´ AOX1 primer) 588 bp (A), 592 bp (B), 593 bp (C)
pPICZD (using the D-Factor primer) 299 bp (A), 303 bp (B), 304 bp (C)

 

If you use the D-factor primer as a PCR primer, you will not see a band with
either GS115 or KM71H. This is because there is no D-factor signal associated
with the chromosomal AOX1 gene.


Sometimes there will be ghost bands appearing in your PCR. These do not seem to be significant as they have not been shown to be a problem.

72
Isolating Total DNA from Pichia, continued

DNA Precipitation 1. Transfer the supernatant from Step 5, page 71, and add 2 volumes of ethanol
to the superna
tant. Incubate at room temperature for 15 minutes.
2. Centrifuge at 10,000 u g for 20 minutes at 4°C.
3. Resuspend the pellet gently in 0.7 ml of TE buffer, pH 7.4 and transfer to a
microcentrifuge tube.
4. Gently extract with an equal volume of phenol:chloroform (1:1 v/v)
followed by an equal volume of chloroform:isoamyl alcohol (24:1). Split the
aqueous layer into two microcentrifuge tubes.
5. Add 1/2 volume of 7.5 M ammonium acetate, pH 7.5, and 2 volumes of
ethanol to each tube. Place on dry ice for 10 minutes or at …20°C for
60 minutes.
6. Centrifuge at 10,000 u g for 20 minutes at 4°C and wash the pellets once with
1 ml of 70% ethanol. Briefly air dry the pellets and resuspend each one in
50 µl of TE buffer, pH 7.5. Determine the concentration of the DNA sample.
You may store the samples separately or combined at …20°C until ready for
use.