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EasySelect™ Pichia Expression Kit (Invitrogen™)

The EasySelect™ Pichia Expression Kit provides all needed components for protein production in the yeast Pichia pastoris. The EasySelect™ Kit contains Pichia yeast strains, expression vectors, and reagents that enable transformation of Pichia cells. Selection with Zeocin™ antibiotic makes it easy to screen for high-copy number transformed variants.

Advantages of the Pichia pastoris expression system:
• Culture Pichia cells as easily as E. coli
• Obtain higher cell density than other microbial expression systems.
• Generate more protein per cell than other microbial expression systems.
• Benefit from eukaryotic protein processing, protein folding, and post-translational modifications.
• Easily scale-up from shake flasks up to industrial-scale fermentation.

A Proven Protein Expression System
Over the past 30 years, Pichia pastoris has been used by labs and industry around the world for producing hundreds of different proteins from many species including human (Ref 1, 2, 3). Pichia pastoris may be grown as easily as bacteria (E. coli) or yeast (Saccharomyces cerevisiae) yet offers many advantages over those systems (Ref 4). Pichia pastoris can grow to much higher cell densities than bacteria or other yeast. Pichia pastoris is capable of growing on methanol as a sole carbon source making scale-up to industrial levels relatively inexpensive.

The Power of the AOX1 Promoter
The EasySelect™ Pichia Expression Kit uses the promoter from the Pichia alcohol oxidase 1 (AOX1) gene to drive production of your protein. Pichia pastoris is capable of growing on methanol as its only carbon source. When grown on methanol as sole carbon source, AOX1 mRNA represents up to 5% of total mRNA and AOX1 enzyme can be up to 30% of cell weight (Ref 5).

The EasySelect™ Pichia Expression system is supplied with the pPICZ and pPICZα vectors. The pPICZ and pPICZα vectors contain a multiple cloning site (MCS) and allow the insertion of your gene of interest under control of the AOX1 promoter. The pPICZ and pPICZα vectors also contain a c-Myc tag for easy detection and a polyhistidine (6xHis) tag for easy detection and purification of your protein.

Since Pichia pastoris naturally secretes few proteins, you can use the extracellular signaling sequence in the pPICZα vectors to force secretion of your protein into the culture medium to simplify down-stream purification.

Get High-Copy Number Integration with Zeocin™ Antibiotic
The EasySelect™ Pichia Expression Kit utilizes the Zeocin™ antibiotic to select for transformed Pichia cells. pPICZ and pPICZα vectors contain the bleomycin gene from Streptoalloteichus hindustanus (Sh ble) that confers resistance to Zeocin™ antibiotic. The pPICZ and pPICZα vectors can be stably integrated into the Pichia genome. By increasing the concentration of Zeocin™ antibiotic, you can easily select for clones with the highest number of integrants and highest protein expression levels.

The EasySelect™ Pichia Expression Kit contains the following components:
• Stab vials of Pichia pastoris and E. coli strains for starting your cultures.
• Solutions for making transformation-competent Pichia cells.
• pPICZ and pPICZα vectors for fusing your gene to the AOX1 promoter.
• Sequencing primers for confirming insertion of your gene in the pPICZ vectors.
• Zeocin™ antibiotic for selecting transformed Pichia cells.

For Research Use Only. Not intended for use in diagnostic procedures.

Related Links
View the product insert for the pPICZ A, B, C vectors (PDF).
View the product insert for the pPICZα A, B, C vectors (PDF).
View the product insert for Zeocin™ antibiotic (PDF).
Learn more about using Pichia pastoris in Pichia Protocols 1st edition available from Invitrogen.
Learn more about other Pichia expression systems from Invitrogen.

References

1. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45-66. [PubMed]
2. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002 Aug;13(4):329-32. [PubMed]
3. Cregg JM. Introduction: distinctions between Pichia pastoris and other expression systems. Methods Mol Biol. 2007;389:1-10. [PubMed]
4. Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T. Expression in the yeast Pichia pastoris. Methods Enzymol. 2009;463:169-89. [PubMed]
5. Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol. 1989 Mar;9(3):1316-23. [PubMed]

Multi-Copy Pichia Expression Kit (Invitrogen™)

The Multi-Copy Pichia Expression Kit contains the pPIC3.5K, pPIC9K, and pAO815 vectors for production and selection of Pichia strains that contain more than one copy of the gene of interest. In many cases, increased copy number has led to increased expression levels.

pPIC9K and pPIC3.5K
The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance gene that confers resistance to Geneticin® Reagent in Pichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin® Reagent. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin® Reagent. The ability to grow in high concentrations of Geneticin® indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome (1). The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K remain intracellular.

pAO815
pAO815 is a Pichia expression vector designed to clone multiple copies of a gene into a single vector. The vector contains a Bgl II site upstream of the 5´ AOX1 gene and a unique BamH I site downstream of the 3´ AOX1 transcription termination (TT) signal. Four steps are required to generate multiple copies of the gene of interest:
1. The gene is cloned into the unique EcoR I site in the vector.
2. The construct is digested with BamH I and Bgl II to release the "expression cassette" containing the AOX1 promoter, gene of interest, and 3´ AOX1 TT.
3. Concatemers of the expression cassette are generated by ligation in vitro.
4. The multiple copies are inserted back into the pAO815 vector and transformed into Pichia.

These vectors are also available separately.

Bac-to-Bac™ HBM TOPO™ Cloning Kit (Gibco™)

Bac-to-Bac® Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli (DH10Bac™) rather than homologous recombination in insect cells. The new pFastBac™ HBM TOPO® vector enables secreted protein expression because it has the honeybee melittin (HBM) secretion signal. The new vector should be strongly considered for the expression of glycoproteins. Glycoproteins cannot be glycosylated in the absence of any secretion signal. In contrast to glycoproteins secreted from mammalian cells, glycoproteins secreted from baculovirus can be easily de-glycosylated in vitro. This is a very important feature in order to crystallize proteins!

The pFastBac™ HBM TOPO® vector has also a C-terminal His-Tag with a TEV cleavage signal to enable easy purification of Histidine fusion proteins on nickel-chelating resins (ProBond™ Purification System) and native proteins with the aid of AcTev™ protease.
The vector uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Mimic™ Sf9, Sf21, and High Five™ cells in Sf-900 II & Sf-900™ III media from Gibco.

Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. This requires a time-consuming plaque assay. Bac-to-Bac®'s pFastBac™ vectors, however, recombines with the parent bacmid in DH10Bac™ E. coli competent cells to form an expression bacmid. Transfect the bacmid into insect cells for fast production of a high titer of pure recombinant baculovirus particles in the very first transfection. You'll save weeks of precious time. Collect a pure P2 baculovirus stock on Day 10 without the necessity of tedious plaques assay.

Expressway™Lumio™ Expression and Detection System, without vector (Invitrogen™)

The Expressway™ Lumio™ Expression and Detection System takes advantage of the Lumio™ recognition sequence, a small, six amino acid sequence (Cys-Cys-Pro-Gly-Cys-Cys). The Lumio™ detection reagent binds the recognition sequence with high specificity and affinity, resulting in a bright fluorescent signal for real-time protein production analysis and immediate in-gel protein detection. In addition, Expressway’s specialized E. coli lysate, derived from a slyD mutant, eliminates nonspecific binding of the Lumio™ Green Detection Reagent to the endogenous SlyD protein and provides optimal background for detection of recombinant proteins (Figure 1). The Lumio™ Green Detection Kit is included in the system for real-time detection of protein synthesis as well as easy in-gel protein detection. The Lumio™ vectors (Figures 2 and 3) also feature:

attR sites for efficient recombination with any attL-flanked Gateway® entry vector
N-terminal Lumio™ tag (pEXP3-DEST vector) with TEV cleavage site for efficient removal of the Lumio™ sequence following purification
C-terminal Lumio™ tag (pEXP4-DEST vector) for easy, and immediate in-gel detection
T7 promoter, ribosome binding site, and T7 terminator optimally spaced for cell-free protein expression

PichiaPink™ Secretion Optimization Kit (Invitrogen™)

The PichiaPink™ Secretion Optimization Kit is a new recombinant protein expression system based on the yeast Pichia pastoris. The PichiaPink™ Secretion Optimization Kit gives you convenient and cost-efficient bioproduction from shake flask to 1000-liter production of your recombinant protein. The PichiaPink™ Secretion Optimization Kit comes with our new Pichia strains, the pPINK-HC and pPINK-LC vectors, and secretion signal sequences for production of your protein.

•   Get a complete kit to get started with protein production in Pichia pastoris.

•   Screen your Pichia transformants rapidly by auxotrophy and color selection.
•   Reduce protein degradation with protease-deficient Pichia pastoris strains.
•   Optimize protein secretion with one of 8 secretion signal sequences.

The PichiaPink™ Secretion Optimization Kit comes with:
•   PichiaPink™ Vector Kit (Cat. No. A11152)
•   PichiaPink™ Secretion Signal Set (Cat. No. A11155)
•   PichiaPink™ Expression Strain Set (Cat. No. A11154)
•   PichiaPink™ Media Kit (Cat. No. A11156)

A New Way to Screen Pichia Transformants
The PichiaPink™ Secretion Optimization Kit uses adenine auxotrophy to select for Pichia transformants. The PichiaPink™ strains have the ade2 genotype and require complementation with the ADE2 gene to grow without adenine. Use of an auxotrophic marker also makes it easier to maintain transformants. No antibiotics are required to maintain your transformants.

In addition, you can screen for Pichia transformants by color. The untransformed PichiaPink™ strains appear as pink colonies while transformed PichiaPink™ strains appear as white colonies (Fig 1).

New Expression Vectors for Pichia
The PichiaPink™ Secretion Optimization Kit comes with the new PichiaPink™ Vector Kit. The kit includes two vectors, pPINK-LC and pPINK-HC. The pPINK vectors are built around the ADE2 gene for complementing the ade2-deficient Pichia strains. Both vectors use the methanol-induced AOX1 promoter to express your protein. The pPINK-LC integrates at low-copy number into the Pichia genome while the pPINK-HC vector integrates with high-copy number.

Both pPINK-LC and pPINK-HC vectors are compatible with the PichiaPink™ Secretion Signal Set. Using a 3-way ligation, you can add one of 8 secretion sequences to your gene. Note: the pPINK vectors express proteins intracellularly by default.

Both the PichiaPink™ Vector Kit (Cat. No. A11152) and PichiaPink™ Secretion Signal Set (Cat. No. A11155) can be ordered separately to refill the PichiaPink™ Secretion Optimization Kit.

Keep Your Proteins Intact
The PichiaPink™ Secretion Optimization Kit comes with the PichiaPink™ Expression Strain Set. This set contains the 4 Pichia pastoris strains for use with the PichiaPink™ Expression System. All 4 strains have the ade2 genotype. In addition, 3 strains include knockouts in 2 protease genes. Protease-deficient strains reduce the need for protease inhibitors when growing your cells and improve the yield of your protein. Test a single protease knockout or the double protease knockout to find the one that works best for you.

You can order the PichiaPink™ Expression Strain Set (Cat. No. A11154) separately to refill the PichiaPink™ Secretion Optimization Kit.

Media Pouches to Get You Started

The PichiaPink™ Media Kit is a set of convenient prepackaged media pouches for use with the PichiaPink™ Secretion Optimization Kit. The PichiaPink™ Media Kit contains media needed to grow PichiaPink™ strains from frozen stocks to cultures ready for transformation and selection. You can order the PichiaPink™ Media Kit (Cat. No. A11156) separately to refill the PichiaPink™ Secretion Optimization Kit.

Why Choose the PichiaPink™ Yeast Expression System?
The PichiaPink™ Yeast Expression System is based on the yeast Pichia pastoris. Advantages of Pichia pastoris include rapid growth, well-defined genetic background, simple media formulation, and easy handling. For over 30 years, Pichia pastoris has been used by labs around the world for producing hundreds of different proteins from many species including human (Ref 1, 2). The PichiaPink™ Yeast Expression System allows convenient and cost-effective protein production from small to large scales.

For information on obtaining a commercial-use license for the PichiaPink™Yeast Expression System, please inquire at outlicensing@lifetech.com.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Related Links
Learn more about the PichiaPink™ Yeast Expression System.
Learn more about our other protein expression systems.

References

1. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45-66. [PubMed]
2. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002 Aug;13(4):329-32. [PubMed]

Bac-to-Bac™ HBM TOPO™ Secreted Expression System (Gibco™)

Bac-to-Bac baculovirus expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli rather than homologous recombination in insect cells. The pFastBac HBM TOPO expression system features:

Expression of secreted proteins—The Bac-to-Bac HBM TOPO vector contains the honeybee melittin (HBM) secretion signal to enable secreted protein expression. Because glycoproteins cannot be glycosylated in the absence of a secretion signal, this vector is recommended for the expression of glycoproteins.

Flexibility—The Bac-to-Bac HBM TOPO vector contains a C-terminal His-tag with a TEV cleavage site to enable easy purification of His-tagged proteins with nickel-chelating resins (such as the Invitrogen ProBond Purification System) and generation of native proteins with the aid of the Invitrogen AcTEV Protease.

Fast cloning, easy screening—Blunt TOPO cloning technology enables cloning of blunt-end PCR products into the pFastBac TOPO vector in five minutes. In addition, the pFastBac TOPO expression kit is supplied with Mach1-T1R E. coli that enable the visualization of colonies eight hours after plating on ampicillin-selective plates due to their faster doubling time compared to other standard cloning strains. Find out more about the advantages of TOPO cloning ›

High transfection efficiency with ExpiFectamine Sf Transfection Reagent—The Bac-to-Bac TOPO expression kits now come with the next-generation ExpiFectamine Sf Transfection Reagent for efficient DNA transfection in insect cells using fast, flexible protocols. Find out more about this reagent ›

Time-saving expression bacmid—With the Bac-to-Bac system, the expression cassette of the pFastBac vector recombines with the parent bacmid in DH10Bac E. coli Competent Cells to form an expression bacmid. The bacmid is then transfected into insect cells for production of recombinant baculovirus particles.

Easy colony screening—The parent bacmid in DH10Bac E. coli contains a segment of the lacZa gene. The lacZa gene is disrupted upon transposition of the expression cassette into the bacmid, allowing for blue/white selection of recombinants for easier identification of recombinant colonies.

High protein expression—The pFastBac vector uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell line such as Sf9, Sf21, and High Five cells.

MAX Efficiency™ Transformation Reagent for Algae (Invitrogen™)

MAX Efficiency® Transformation Reagent for Algae is a buffer that enhances transformation efficiency for multiple strains of Chlamydomonas species. One of the biggest hurdles in research and development with Chlamydomonas has been the introduction of exogenous DNA into Chlamydomonas strains due to rigid cell walls. Methods such as glass bead agitation, electroporation, and microparticle bombardment are available but often result in low transformation efficiency.

MAX Efficiency® Transformation Reagent for Algae increases the permeability of the Chlamydomonas cell wall and facilitates increased delivery of DNA into the cell nucleus by electroporation. Simply grow the Chlamydomonas cells to early log phase, harvest by centrifugation, and wash twice with MAX Efficiency® Transformation Reagent prior to electroporation. To date, we have seen >200-fold increase in transformation efficiency over previously recommended conditions in 10 different Chlamydomonas strains, including wild type and mutants, using circular or linear DNA, as well as PCR fragments.

• Obtain >1000 transformants/µg DNA for cell wall(+) strains
• Obtain >100 transformants/µg DNA for cell wall(-) strains
• Transform circular or linear DNA or PCR fragments

Jump-In™ CHO-K1 Kit

The Jump-In™ CHO-K1 Kit allows the targeted integration of genetic material into a specific pre-engineered R4 site in the Jump-In™ CHO-K1 cell line, to create an isogenic stable cell line with less effort and in less time than traditional cell engineering methods.

The high retargeting efficiency, made possible by the R4 sites in the CHO-K1 cell line, allows the use of the isogenic pool for additional experiments without the need for clonal selection. Alternatively, the high retargeting efficiency allows for the easy selection of a positive stable clone expressing your gene of interest.

The Jump-In™ CHO-K1 Kit Lets You:

• Quickly and efficiently develop stably engineered isogenic cell pools in about half the time compared to traditional cell engineering methods
• Utilize isogenic expression from a defined genomic locus as the ideal solution for comparative analysis of gene families, isoforms, or orthologs
• Generate multiple cell lines in parallel using the simplified work flow
• Easily access the technology without complicated licenses or restrictions to interpret

Save Time with Rapid and Efficient Generation of Engineered Cell Lines
With the Jump-In™ CHO-K1 Kit you can generate functional cell pools in as little as 2 weeks without laborious clone isolation and analysis, and the streamlined protocol makes it easier to generate several cell lines at the same time. Even generation of clonal cell lines can be done with reduced time and effort due to the high percentage of positive clones. In addition, the Jump-In™ technology gives you the freedom to generate an unlimited number of cell lines without restrictive licensing requirements.

Expand Your Experimental Capabilities
The Jump-In™ CHO-K1 Kit is the ideal solution for cells and assays where transient engineering technologies are problematic, as well as for difficult to The kit also provides a convenient way to create target panels of gene families, isoforms, or orthologs. Genes coding for large proteins or multi-unit proteins are not a problem since the Gateway® destination vectors accept large inserts.

The kit includes:
• pJTI™ R4 Dest CMV pA vector (100 µg)
• pJTI™ R4 Int vector (100 µg)
• Jump-In™ CHO-K1 Cells (2 vials @ 1 ml each)

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Expi293™ GnTI- Expression System Kit (Gibco™)

The Expi293 GnTI- Expression System Kit is part of the Expi293 platform for rapid, high-yield transient protein expression from mammalian 293 cells. The system kit is centered on engineered Expi293F cells that do not have N-acetylglucosaminyltransferase I (GnTI) activity and therefore lack complex N-glycans. The Expi293F GnTI- cell line was established by the selective knockout of the GnTI gene in parental Expi293F cells. This cell line is a powerful tool for the high-yield expression of homogeneously glycosylated recombinant proteins.

Expi293 GnTI- Expression System Kit features include:
• Homogeneous N-glycosylation of expressed proteins
• Equivalent protein yields to parental Expi293F cells—up to 1 g/L
• Significantly higher protein yields than other HEK293 GnTI- cell line alternatives
• Suspension, high-density culture of Expi293F GnTI- cells in Expi293 Expression Medium
• High-efficiency transient transfection using ExpiFectamine 293 reagent in combination with specialized transfection enhancers

The components included in the Expi293 GnTI- Expression System Kit are: one vial of frozen Expi293F GnTI- cells, 1 L of Expi293 Expression Medium, one ExpiFectamine 293 transfection kit sufficient to transfect 1 L of culture, 100 mL Opti-Plex Complexation Buffer, one PNGase F Glycan Cleavage kit, and the pRABBIT IgG IRES-EmGFP positive control vector. All components of the Expi293 GnTI- Expression System are also available for individual purchase.

Expressway™ Maxi Cell-Free E. coli Expression System with pEXP5-NT/TOPO™ and pEXP5-CT/TOPO™ vectors (Invitrogen™)

The Expressway™ Maxi Cell-Free E. coli Expression System uses an efficient, coupled transcription/translation reaction to produce milligram quantities of soluble, functionally active protein in 4-6 hours. The procedure can be performed in a single reaction tube and is easily scalable without the need for specialized equipment. The TOPO TA Cloning® expression vectors (5-min cloning with 95% efficiency) are provided for optimal expression results. In addition to all the advantages of an open expression system, Expressway™ Maxi technology provides a means to produce high levels of recombinant protein that may be easily detected and purified for various downstream applications.

Milligram level protein production within 4-6 hours
The key to the Expressway™ Maxi technology is the unique formulation of the Feed Buffer that boosts the reaction productivity so that more than one milligram of protein is produced in a 2-ml reaction within 4-6 hours (Figure 1). The formulation of the lysate enables protein synthesis using either circular or linear templates.

High-throughput (HTP) compatible
This system is also designed for HTP expression. One kit is good for 200 x 50-reactions, which can be accommodated by 2 x 96-well plates. Without feed buffer, such reactions need only two hour’s time. Once a positive expression is detected, scale-up protein production can be performed with the large Expressway™ cell-free format or in E. coli cell-based systems.

Optimized vectors
pEXP5 TOPO® TA vectors are optimized for use in the Expressway™ Milligram system (Figure 2). They are designed to minimize additional amino acid sequences that may interfere with native protein folding and functionality. Features of these vectors include:

• pEXP5-NT/TOPO® expression vector provides N-terminal histidine tag and TEV protease recognition site

• pEXP5-CT/TOPO® expression vector can be used for expressing proteins with a C-terminal histidine tag or for native protein expression by introducing a stop codon at the end of your gene of interest

Simple and fast procedure
The Expressway™ Maxi Cell-Free Expression System provides all the components needed for optimal cell-free protein production. The system includes an E. coli extract, IVPS reaction buffer, Feed Buffer, T7 enzyme mix, 19 amino acid mix, and individual tubes of methionine. To start a 2-ml reaction, mix the reaction buffer, 19 amino acid mix, your choice of labeled or unlabeled methionine and cysteine, T7 enzyme mix, and your DNA template (with T7 promoter) with the E. coli extract. After a 30-minute incubation, add the Feed Buffer. Milligram-levels of active protein will be synthesized within 4-6 hours. To perform HTP expression, simply premix all the component and aliquot into a HTP format.

MembranePro™ Functional Protein Expression Kit (Invitrogen™)

For customers studying GPCRs and other membrane proteins, MembranePro™ is the ready-to-use system that delivers enriched, functional membrane proteins efficiently and reliably. Benefits of MembranePro™ include:

• Proteins are displayed on human (or other mammalian) cell membranes
• Cellular quality control and mammalian posttranslational processing helps produce functional protein
• Proteins bud off as lipoparticles and are easily collected from the culture medium
• Lipoparticles are enriched with expressed receptors

MembranePro™ workflow is less labor intensive in comparison to traditional production of membrane fractions. Briefly, the MembranePro™ protocol allows you to:

1. Clone your gene of interest using a TOPO® vector
2. Transfect with Lipofectamine™ 2000 and MembranePro™ Reagent into 293FT cells
3. Harvest lipoparticles 48h post-transfection, concentrate with MembranePro™ Precipitation Mix
4. Store lipoparticles at -80 C or proceed to downstream assays

MembranePro™ products are offered in two configurations – as an expression kit (TOPO® vector, Lipofectamine™ 2000, MembranePro™ Precipitation Mix, MembranePro™ Reagent, 293FT cells) and as a support kit (Lipofectamine™ 2000, MembranePro™ Precipitation Mix, MembranePro™ Reagent). The expression kit is only available in a 10 reaction size; the support kit is available in 10, 60 and 600 reaction sizes.

PichiaPink™ Secreted Protein Kit (Invitrogen™)

The PichiaPink™ Secreted Protein Expression Kit is a new recombinant protein expression system based on the yeast Pichia pastoris. The PichiaPink™ Secreted Protein Expression Kit gives you convenient and cost-efficient bioproduction from shake flask to 1000-liter production of your recombinant protein. The PichiaPink™ Secreted Protein Expression Kit comes with our new Pichia strains, the pPINK-HC and pPINK-LC vectors, and secretion signal sequences for production of your protein.
  Get started with secreted protein production in Pichia pastoris.
•   Screen your Pichia transformants rapidly by auxotrophy and color selection.
•   Reduce protein degradation with protease-deficient Pichia pastoris strains.

The PichiaPink™ Secreted Protein Expression Kit comes with:
•   PichiaPink™ Secreted Protein Vector Kit (Cat. No. A11153)
•   PichiaPink™ Expression Strain Set (Cat. No. A11154)
•   PichiaPink™ Media Kit (Cat. No. A11156)

A New Way to Screen Pichia Transformants
The PichiaPink™ Secreted Protein Expression Kit uses adenine auxotrophy to select for Pichia transformants. The PichiaPink™ strains have the ade2 genotype and require complementation with the ADE2 gene to grow without adenine. Use of an auxotrophic marker also makes it easier to maintain transformants. No antibiotics are required to maintain your transformants.

In addition, you can screen for Pichia transformants by color. The untransformed PichiaPink™ strains appear as pink colonies while transformed PichiaPink™ strains appear as white colonies (Fig 1).

Secrete Your Proteins with the pPINKα-HC Vector

The PichiaPink™ Secreted Protein Expression Kit comes with the pPINKα-HC vector. The pPINKα-HC vector has the α-mating factor signal sequence built-in and allows your protein to be secreted into the media. Secreting your protein into the media makes harvesting and purifying your protein easier. The pPINKα-HC vector is built around the ADE2 gene for complementing the ade2-deficient Pichia strains. This vector uses the methanol-induced AOX1 promoter to express your protein. Further, the pPINKα-HC vector is designed to be integrated at high-copy-number in your Pichia transformants.

The PichiaPink™ Secreted Protein Vector Kit (Cat. No. A11153) can be ordered separately to refill the PichiaPink™ Secreted Protein Expression Kit.

Keep Your Proteins Intact
The PichiaPink™ Secreted Protein Expression Kit comes with the PichiaPink™ Expression Strain Set. This set contains the 4 Pichia pastoris strains for use with the PichiaPink™ Yeast Expression System. All 4 strains have the ade2 genotype. In addition, 3 strains include knockouts in 2 protease genes. Protease-deficient strains reduce the need for protease inhibitors when growing your cells and improve the yield of your protein. Test a single protease knockout or the double protease knockout to find the one that works best for you.

You can order the PichiaPink™ Expression Strain Set (Cat. No. A11154) separately to refill the PichiaPink™ Secreted Protein Expression Kit .

Media Pouches to Get You Started

The PichiaPink™ Media Kit is a set of convenient prepackaged media pouches for use with the PichiaPink™ Secreted Protein Expression Kit. The PichiaPink™ Media Kit contains media needed to grow PichiaPink™ strains from frozen stocks to cultures ready for transformation and selection. You can order the PichiaPink™ Media Kit (Cat. No. A11156) separately to refill the PichiaPink™ Secreted Protein Expression Kit.

Why Choose the PichiaPink™ Yeast Expression System?
The PichiaPink™ Yeast Expression System is based on the yeast Pichia pastoris. Advantages of Pichia pastoris include rapid growth, well-defined genetic background, simple media formulation, and easy handling. For over 30 years, Pichia pastoris has been used by labs around the world for producing hundreds of different proteins from many species including human (Ref 1, 2). The PichiaPink™ Yeast Expression System allows convenient and cost-effective protein production from small to large scales.

For information on obtaining a commercial-use license for the PichiaPink™Yeast Expression System, please inquire at outlicensing@lifetech.com.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Related Links
Learn more about the PichiaPink™ Yeast Expression System.
Learn more about our other protein expression systems.

References

1. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45-66. [PubMed]
2. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002 Aug;13(4):329-32. [PubMed]

pTrcHis2 TOPO™ TA Expression Kit (Invitrogen™)

The pTrcHis- and pTrcHis2-TOPO® TA Expression Kits allow fast, efficient cloning of PCR products into a prokaryotic expression vector for high-level, regulated expression from the trc promoter. The vector in each kit, pTrcHis-TOPO® or pTrcHis2-TOPO®, includes a minicistron element for enhanced translation efficiency of eukaryotic proteins in E. coli. Both vectors are provided linearized and activated with topoisomerase I for 5-minute TOPO® Cloning with >85% cloning efficiency. To simplify protein purification and detection, the pTrcHis-TOPO® and pTrcHis2-TOPO® vectors include the following features:

pTrcHis-TOPO®

N-terminal Xpress™ epitope for detection with an Anti-Xpress™ Antibody
• N-terminal polyhistidine (6xHis) tag for purification using nickelchelating resin and detection with an Anti-HisG Antibody
• Enterokinase cleavage site for efficient removal of N-terminal fusion tags

pTrcHis2-TOPO®
C-terminal c-myc epitope for detection with an Anti-myc Antibody
• C-terminal polyhistidine (6xHis) tag for purification using nickel-chelating resin and detection with an Anti-His(C-term) Antibody

Champion™ pET100 Directional TOPO™ Expression Kit with BL21 Star™ (DE3) One Shot™ Chemically Competent E. coli (Invitrogen™)

The Champion™ pET Expression System yields the highest-level protein production in E. coli. During expression, your protein of interest can reach levels greater than 50 percent of total cellular protein. Based on T7 expression vectors originally developed by Studier and colleagues (1-3), high-level expression is achieved because the T7 RNA polymerase is more processive than native E. coliRNA polymerase and is dedicated to the transcription of your gene of interest. Protein production is further enhanced in the system by the expression strain BL21 Star™ E. coli, which significantly improves the stability of mRNA transcripts and increases protein expression up to ten-fold.

Simplified, efficient Directional TOPO® cloning allows you to quickly enter a Champion™ pET Expression vector. These kits feature linearized, topoisomerase I-activated Champion™ pET expression vectors for five-minute directional cloning. Directional TOPO® Cloning technology facilitates gene expression because:

• A proofreading enzyme is used for PCR, resulting in fewer errors in cloned genes
• Greater than 90% of the clones are in the correct orientation for gene expression, reducing the time spent on colony screening

Seven Champion™ pET Directional TOPO® Expression Vectors are available (Figure 1 and Table 1): Each vector carries a T7lac promoter for high-level expression. Flexible options for simplifying protein detection, cleaving purification tags, selecting plasmid carrying clones, and/or improving protein yields are available.
With the Champion™ pET Directional TOPO® vectors you can expect highest-level protein production. Figure 2 shows expression of the lacZ gene in Champion™ pET Directional TOPO® vectors. Figure 3 demonstrates efficient cleavage using TEV protease of the N-terminal tag of a β-galactosidase fusion protein expressed from pET151/D-TOPO®.

ExpiSf™ Expression System Starter Kit (Gibco™)

The ExpiSf Expression System is a complete chemically-defined baculovirus-insect cell protein expression system that delivers superior yields and consistent performance run after run using a fast, streamlined workflow.

The ExpiSf Expression System offers:
• A chemically defined, yeastolate-free, animal origin-free medium for consistent cell growth and protein expression
• Integrated components that work in concert to deliver three times higher protein yields compared to traditional insect expression platforms
• Robust production of high-titer, high-quality P0 recombinant baculovirus in suspension culture format without the need for further virus amplification
• A fast, simplified workflow that allows you to obtain protein in as little as 6–10 days; half the time of traditional protocols
• Scalable expression that can be applied to cultures from 24-deep well plates to >3 L shake flasks

The components included in the ExpiSf Expression System Starter Kit are: 2 vials of frozen ExpiSf9 Cells, 1 liter of ExpiSf CD Medium, ExpiSf Enhancer sufficient for 1 liter of culture, 1 mL ExpiFectamine Sf Transfection Reagent, 500 mL Opti-MEM Reduced Serum Medium, 0.5 mL MAX Efficiency DH10Bac Competent Cells, 1 Bac-to-Bac Vector Kit containing the pFastBac 1 Expression Vector and pFastBac 1-Gus Positive Control Vector. All components of the ExpiSf Expression System are also available for purchase separately.