Shop All Protein Expression Kits

MembranePro™ Functional Protein Support 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™ 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]

ExpiCHO™ Expression System Kit (Gibco™)

The ExpiCHO™ Expression System Kit combines the power of rapid, ultra–high-yield transient protein production in suspension culture with the benefit of expression in Chinese hamster ovary (CHO) cells. It is based on high-density culture of ExpiCHO-S™ cells in ExpiCHO™ Expression Medium. Transient expression is powered by ExpiFectamine™ CHO transfection reagent, designed specifically for high-density CHO suspension culture, in combination with a specialized transfection enhancer and culture feed. All components work together synergistically to generate 2- to 10-fold higher protein yields than the HEK293-based Expi293™ Expression System Kit and 20- to 150-fold higher yields than the FreeStyle™ MAX CHO Expression System. Expression yields of 1 to 3 g per L of transfected culture have been demonstrated for some antibody and non-antibody proteins.

The ExpiCHO Expression System offers:

• Milligram to multigram yields of recombinant protein per liter of culture in 5 to 14 days post-transfection, which are frequently higher than transient 293-based systems
• Cost-effective transient expression in CHO cells, allowing scientists developing protein biotherapeutics to work with CHO-expressed proteins from start to finish in the drug development process
• A robust alternative mammalian transient expression host, allowing for production of proteins that are difficult to express in HEK293 cells
• Flexible, reliable culture and transfection protocols with multiple options that readily fit into current transient expression workflows
• Scalable expression for cultures from <1 mL to >10 L, thus generating the amount of protein needed for your application

The components included in the ExpiCHO Expression System Kit are: 2 vials of frozen ExpiCHO-S cells, 1 L of ExpiCHO Expression Medium, 1 ExpiFectamine CHO Transfection Kit sufficient to transfect 1 L of culture, OptiPRO™ SFM, and an antibody-expressing positive control vector. All components of the ExpiCHO Expression System Kit are also available for purchase separately. All components of the system are animal origin–free.

Freedom™ CHO-S™ Kit (Gibco™)

Gibco® Freedom® CHO-S® Kit, co-developed with ProBioGen AG, is an easy-to-use, beginning-to-end product, for cloning and expression of recombinant proteins in Chinese Hamster Ovary (CHO)-derived suspension culture. The Gibco® Freedom® CHO-S® Kit includes components for transfection, expression, clone creation, and stable cell line selection, all in a conveniently packaged kit. The Gibco® Freedom® CHO-S® Kit also delivers high titers, and offers simplified commercial licensing options.

• Flexible commercial licensing without the need for royalties
• Ability to achieve IgG titers over 3g/L
• Complete workflow that can take your gene of interest from transfection to lead clone typically in 5 months
• Convenient packaging, for simplified ordering and storage

Commercial Licensing that is Simple, Flexible and Without Royalties
Multiple companies provide CHO (Chinese Hamster Ovary) stable cell line development services and products that require annual fees during development of your therapeutic protein. In addition, royalties may be required on the sales of your therapeutic protein upon commercialization. Gibco® Freedom® Kits provide you with the tools that enable you or your CRO provider to develop your stable cell line for just the price of the kit itself. Furthermore, as you move towards commercial use, a flexible license structure is available without the burden of royalties and works to keep it as simple as a onetime payment (Fig 1). For more information regarding licensing, email us at outlicensing@lifetech.com.

Industry Standard Production Levels
Current bioprocess industry standards to achieve manufacturing efficiencies for IgG type molecule production with stable CHO cell lines in bioreactors is 2-3g/L or better. We have demonstrated that the Gibco® Freedom® CHO-S® Kit can consistently generate IgG producing scalable clones (stable out to 60 population doublings), that meet this standard of productivity (Fig 2).

Complete, Fast, and Efficient Workflow
The Gibco® Freedom® CHO-S® Kit’s complete, proven, and fully integrated workflow allows you to move from transfection to lead clone typically in 5 months, with reduced hands on time, and reduced Fulltime Equivalent (FTE) requirements (Fig 3). The entire workflow is performed in our CD FortiCHO™ medium, a chemically defined, animal origin free (AOF) medium. The kit also offers the following advantages:

• Cloning of one or two genes that encode your protein(s) of interest
• High efficiency transfection of DNA into CHO-S® Cells (cGMP-banked)
• Included flash drive with loaded manual

New Convenient Packaging
To free you to focus on the creation of your stable cell line rather than on figuring out where and when all the components of your experiment are arriving, Freedom® CHO-S® Kit introduces the smART™ tote packaging system (shown in above product photos). All components for the creation of your expression cell line are delivered in soft cooler totes along with a parts flyer, and a flash drive containing the manual. Upon delivery you have all the information needed for correct storage and use of components consolidated in the two smART™ tote shipment coolers.

Access to Gibco® Freedom® Kit Support
If any specific support is needed beyond the manual, please contact your account manager to obtain a Process Science Manager consultation. In addition, one of our support scientists may check in with you to ensure that your experience with the kit is as successful as possible. For further assistance, please email our support team at GibcoServices@thermofisher.com.

Champion™ pET200 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®.

MembraneMax™ HN Protein Expression Kit (Invitrogen™)

MembraneMax™ HN Protein Expression Kits delivers high yields of soluble (dispersed) membrane proteins using the MembraneMax™ reagent, which is an planar phospholipid membrane bilayer surrounded by a scaffold protein (also called a nanolipoprotein particle or NLP) that also offers convenient His-tag purification. Combining this technology with a scalable cell-free expression E. coli extract delivers microgram to milligram quantities of your membrane protein specifically for high throughput expression screening, expression of membrane proteins that would otherwise be toxic in cell-based systems and functional or activity assays. With high uniformity and consistent structure, MembraneMax reagent minimizes formation of membrane protein aggregates or clumping often caused by inconsistent size, shape and structure typical observed with microsomes or micelles. The MembraneMax™ Protein Expression kits contain all the necessary reagents for successful expression of your membrane protein.
Key Advantages of the MembraneMax™ Protein Expression Kits:
• Optimized cell-free expression delivers high yield of membrane protein from nanogram to milligram quantities
• MembraneMax™ reagent enables a monodispersed population of soluble membrane protein-NLP complexes
• Convenient His-tag allows for simple purification of native or un-tagged membrane proteins
• Expression format amenable to high-throughput protein synthesis for screening and expression of toxic proteins
• Production of a homogeneous population of your membrane protein
• Convenient kit format includes the necessary reagents for protein expression—simply add your gene of interest to get started

NativePure™ Lentiviral Expression and Affinity Purification System

The NativePure™ Lentiviral Expression and Affinity Purification System provides an efficient method for lentiviral delivery of your gene of interest into cells, and the purification and analysis of native protein complexes. The NativePure™ Lentiviral Gateway® Vectors allow N- or C- terminal fusion of your recombinant protein of interest to the capTEV± affinity purification tag.

The Lentiviral Expression and Affinity Purification System provides:
• Gateway® vectors designed to express your protein of interest in multiple expression systems
• Lentiviral vectors with the capTEV™ Tag that allow in vivo biotinylation of your protein of interest
• Streamlined purification protocols with Streptavidin Agarose

Contents:
The NativePure™ Lentiviral Expression and Affinity Purification System includes the NativePure™ Lentiviral Gateway® Vector Kit, the NativePure™ Lentiviral Expression Kit, and the NativePure™ Lentiviral Affinity Purification Kit. Each kit is also available separately.

The NativePure™ Lentiviral Gateway® Vector Kit consists of the pLenti6/capTEV™-NT-DEST1 (N-terminal vector), pLenti6/capTEV™-CT-DEST (C-terminal vector), and pLenti6/capTEV™-CT-GW/ARP2 (C-terminal) control vector.

The NativePure™ Lentiviral Expression Kit contains the NativePure™ Lentiviral Gateway® Vector Kit, the ViraPower™ Bsd Lentiviral Support Kit, 293FT Cell Line, One Shot® Stbl3™ Chemically Competent E. coli, and Gateway® LR Clonase® Enzyme Mix.
The NativePure™ Lentiviral Affinity Purification Kit contains the NativePure™ Lentiviral Gateway® Vector Kit, Streptavidin Agarose, NativePure™ 5X Lysis/Binding Buffer, NativePure™ 10X TEV Cleavage Buffer, 100 mM DTT (Dithiothreitol), 10% NP40, AcTEV™ Protease, NativePure™ Columns, and NativePure™ Concentrators.

Store the 293FT Cell Line in liquid nitrogen and the One Shot® Stbl3™ Chemically Competent E. coli at -80°C.

Store Streptavidin Agarose, 10% NP-40, NativePure± 5X Lysis/Binding Buffer, NativePure± 10X TEV Cleavage Buffer, Lipofectamine™ 2000, NativePure± Columns, and NativePure± Concentrators at +4°C.

Store the NativePure™ Lentiviral Gateway® Vector Kit, AcTEV± Protease, 100 mM DTT, Gateway® LR Clonase® Enzyme Mix, ViraPower™ packaging Mix, and Blasticidin at -20°C. All components are guaranteed stable for 6 months when properly stored.

Champion™ pET151 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®.

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]

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

Expi293™ MembranePro™ Expression System (Gibco™)

For researchers studying G protein–coupled receptors (GPCRs) and other membrane proteins, the Expi293™ MembranePro™ Expression System delivers enriched, functional membrane proteins with high efficiency, scalability and reliability for a wide variety of low or high throughput applications.

Features of the Expi293™ MembranePro™ Expression System include:
• Efficient—more than 20-fold increase in membrane protein yield compared to the adherent culture system
• Scalable—easy scale-up without the need for multiple flasks
• Easy to use—no enzymatic or mechanical dissociation required

In addition, benefits of MembranePro™ technology include:
• Proteins are displayed on human (or other mammalian) cell membranes
• Cellular quality control and mammalian post-translational processing helps produce functional protein
• Proteins bud off as lipoparticles and are easily collected from the culture medium
• Lipoparticles are enriched with expressed receptors

With the less labor-intensive Expi293™ MembranePro™ Expression System protocol, you will:
1. Clone your gene of interest using a TOPO® vector
2. Transfect a susupension culture of Expi293F™ cells using Expifectamine™ 293 transfection reagent
3. Harvest lipoparticles 48 hrs after addition of enhancers using MembranePro™ Precipitation Mix
4. Store lipoparticles at -80 C or proceed to downstream assays

Note: Exi293F™ Cells (A14527) and a pEF6 V5-His TOPO® Expression Kit (K9610-20) are required for use of this system and must be purchased separately when needed.

pBAD/TOPO™ ThioFusion™ Expression Kit (Invitrogen™)

The pBAD/TOPO® ThioFusion™ Expression Kit is designed for one-step cloning and regulated expression of thioredoxin fusion proteins in E. coli(Figure 1). The pBAD/Thio-TOPO® vector encodes His-Patch thioredoxin as an N-terminal fusion partner. The thioredoxin fusion can significantly increase the solubility of many difficult-to-express proteins and improve the yield of protein production. The vector includes the following additional features (Figure 2):

• The araBAD promoter for tightly regulated expression in E. coli I-activated vector for 5-minute TOPO® Cloning of Taq-amplified PCR products
• C-terminal polyhistidine (6xHis) tag for purification with nickel-chelating resin and detection with an Anti-His(C-term) Antibody
• C-terminal V5 epitope for detection with an Anti-V5 Antibody
• Enterokinase cleavage site for efficient cleavage of N-terminal fusion tag with EKMax™ Enterokinase

GeneArt™ Synechococcus Protein Expression Vector (Invitrogen™)

The GeneArt™ Synechococcus Protein Expression Vector is our second-generation Synechococcus protein expression vector optimized for relative high-level expression in algae and with dual protein tags for detection and/or purification of your gene of interest. The kit includes expression vector and easy-to-follow protocols. Our Gibco™ BG-11 Media, sold separately, is optimized for the growth and maintenance of select Cyanobacteria including Synechococcus elongatus, available from the ATCC collection.

• Express >10% total soluble protein of your gene of interest
• Detect and purify your gene of interest with 6His-TEV and/or V5-His epitope tags
• Compatible with seamless assembly for creation of constructs

A specific decided expression vector
The GeneArt Synechococcus Protein Expression Vector is designed to provide constitutive expression in Synechococcus elongatus. Expression is controlled by promoter psbA1, a ribosome binding site (RBS), and optional initiation and stop codons depending on gene location. The vector provides purification and detection tags at the N- and C-termini: an N-terminal polyhistidine tag with a TEV recognition site for optional cleavage and a C-terminal V5 epitope followed by a polyhistidine tag. Two MCS sites provide the flexibility to attach either, both, or no tags toyour protein. Our pSyn_6 vector is also compatible with seamless cloning, an optional cloning method that results in no extra sequences in your final construct.

Features of the vector include:
• Strong constitutive promoter, psbA1, for robust expression of recombinant gene of interest
• Ribosome binding site (RBS) for improved expression of some genes
• Targeted integration of your gene of interest into the Synechococcus elongatus genome
• N-terminal 6His-TEV and C-terminal V5-6His epitope tags
• Spectinomycin-resistance gene for selection in Synechococcus
• Flexible, multiple cloning site vector

>80% integration efficiency
The transformation of Synechococcus elongatus PCC7942 relies on homologous recombination between the cell’s chromosome and exogenous DNA that is not autonomously replicating and contains sequences homologous to the chromosome. The location of integration into the chromosome (neutral site, NS1) was developed as a cloning locus (Clerico et al., 2007), since it can be disrupted without any aberrant phenotype, thus allowing recombination of ectopic sequences. When transformed with vectors containing an antibiotic resistance cassette and neutral site sequences, a double homologous recombination event occurs between the neutral site vector and the Synechococcus elongatus chromosome. The selective marker (spectinomycin) and the gene of interest driven by a promoter are inserted into the neutral site and the vector backbone (pUC) is lost, allowing the expression of recombinant genes in Synechococcus elongatus PCC 7942.

Gibco BG-11 Media—optimized for cyanobacteria
Gibco BG-11 Media, available separately, is optimized for the growth and maintenance of select Cyanobacteria including Synechococcus elongatus. The 1X formulation lets you avoid laborious media preparation steps. Award-winning bottle design is easier to use in the biosafety cabinet, minimizes the risk of contamination, and helps you perform cell culture more consistently. Superior packaging and quality, greater reliability, and improved consistency in Cyanobacteria culture results in better overall efficiency and more robust data.

Jump In™ T-REx™ CHO-K1 Kit

The Jump-In™ T-REx™ 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 inducible isogenic stable cell line with less effort and in less time than traditional cell engineering methods. These cells stably express the tetracycline repressor protein, allowing for inducible expression of your retargeted gene of interest upon the addition of doxycycline.

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™ T-REx™ 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
• Inducibly express your gene of interest using doxycycline
• 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™ T-REx™ CHO-K1 Kit you can generate functional cell pools in as little as 2 weeks without laborious clone isolation and analysis, and the streamlined workflow 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™ T-REx™ CHO-K1 Kit allows for inducible expression of your toxic or unstable gene of interest and is the ideal solution for targets and assays where transient engineering technologies are problematic. 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:

Jump-In™ T-REx™ CHO-K1 Cells (2 vials @ 1 ml each)
pJTI™ R4 Dest CMV TO pA Vector (100 µg)--A Gateway® Destination vector that drives inducible expression of your gene of interest under control of the strong CMV promoter by inclusion of the Tet-Operon.
pJTI™ R4 Int Vector (100 µg)--Expresses the R4 integrase to facilitate the retargeting of your gene of interest into the R4 site.

Sold separately:

pJTI™ R4 CMV-TO MCS pA Vector--A multi-site cloning vector that drives inducible expression of your gene of interest under control of the strong CMV promoter by inclusion of the Tet-Operon.
pJTI™ R4 EXP CMV-TO EmGFP pA Vector--A control vector with inducible expression of GFP under control of the strong CMV promoter by inclusion of the Tet-Operon.

1-Step Human Coupled IVT Kit - DNA (Thermo Scientific™)

Thermo Scientific 1-Step Human In Vitro Protein Expression Kits enable the translation and post-transcriptional modification of full-length proteins from mRNA or plasmid templates with yields of up to 100 µg/mL per reaction.

The Human IVT Kits are unique HeLa cell lysate-based protein expression systems for in vitro translation or coupled transcription/translation reactions. Protein expression is performed in a single 90-minute reaction that can be extended for up to 6 hours with continued protein production up to 100 µg/mL when combined with the optimized pT7CFE1 Expression Vector. The Human IVT Kits can express functional enzymes, phosphoproteins, glycoproteins and membrane proteins for immediate use in studying protein interactions, performing rapid mutational analysis and measuring activity.

Features of the 1-Step Human Coupled IVT Kit:

Functional—uses the human translational machinery to express active proteins
Convenient—perform transcription and translation in a single step
High performance—greater yields compared to rabbit reticulocyte in vitro translation
Reliable—express proteins that fail in rabbit reticulocyte systems

Better Than Traditional Methods:
• HeLa cell-free extracts are capable of expressing proteins with post-translational modifications
• Accurate translation delivers full-length protein compatible with downstream applications
• Protein translation is optimized with EMCV IRES element and other mRNA stabilizing elements

Includes:
• Complete kits contain HeLa cell lysate, accessory proteins, reaction mix, nuclease-free water, expression vector and a GFP-positive control vector

Requires:
• 30°C incubator or water bath

Applications:
• Express proteins to measure enzyme activity
• Perform mutational analysis
• Express protein for use in gel mobility shift assays
• Perform co-immunoprecipitation
• Express cytotoxic proteins
• Perform unnatural amino acid labeling

The 1-Step Coupled Human IVT Kit for DNA is a cell-free system using the cellular transcription and translation machinery from a modified HeLa cell line. The procedure is quick and easy and is an effective alternative to other protein expression methods. Simply add an appropriate expression construct to a mixture of HeLa cell lysate, Accessory Proteins, Reaction Mix and incubate at 30°C for 90 minutes to overnight.

The 1-Step Human IVT Kit for mRNA also uses the human protein translation machinery to produce functional protein. This kit is recommended for translation of mRNA transcripts containing an EMCV IRES element and mRNA stabilizing features designed into the pT7CFE1 expression vector.

The human in vitro translation system is robust and will express proteins from a variety of species including mammals, bacteria and protozoa. Benchmarking shows that the expression levels of functional proteins such as luciferase are much higher in the human system compared to either rabbit reticulocyte-based or E. coli-based systems. Furthermore, proteins expressed with the human in vitro translation system are not contaminated with substances that can interfere with downstream applications.

More Product Data
Protocol for in vitro protein expression using mRNA templates
Glycoprotein expression in a human IVT system