Mammalian Cell Expression System

Over the past twenty years, the production of recombinant proteins (r-proteins) has become increasingly useful with regards to biopharmaceutical products with medical applications, particularly purified r-proteins suitable for structure-function analysis. Most r-proteins applied in this capacity are complex molecules that need to be modified post-translationally for full bioactivity and native conformation. In this respect, cultivated mammalian cells produce superior r-proteins because they possess the proper machinery for protein-folding and post-translational modification. GenScript recommends its mammalian expression service for cases in which the folding of the proteins is crucial to the customer's intended application and cases in which the internal machinery of E.coli is unable to express the protein in sufficient time or quality. Our protein expression team can produce milligram-levels of protein from mammalian cells in less than four weeks. Researchers can then follow up on promising leads in an expeditious manner.

Advantages of Mammalian Cell Expression:

Ability to execute special post-translational modifications and proper folding
Stable and rapid transient protein expression
Powerful promoter (CMV, SV40) to regulate protein expression
High expression levels
Large-scale protein production

Services Included:

I. Vector construction and improvement

For either stable or transient expression, GenScript can clone the customer's cDNA construct into either the customer's mammalian expression vector or one provided by GenScript. Upon request, GenScript will add secretory signals or tags to each expression vector to facilitate the eventual recovery of the protein through affinity chromatography. For more complex projects, such as those requiring specific post-translational modifications, the formation of multimeric complexes, the formation of specific disulphide bridges within or between peptides, or special glycosylation, GenScript recommends its custom vectors. We can also label the desired gene, gene fragment, or fusion construct with a fluorescent marker, such as GFP. In addition, we can generate and identify high-productivity stable cell lines, suitable for scale-up, cGMP production, or both.

II. Stable gene expression (SGE) for long-term production purposes

The entire work, from transfection, selection, and isolation of clonal cell populations through the evaluation of primary clones for productivity and adaptability for growth in serum-free suspension culture, offers long-term production stability and scalability. Upon request, GenScript will apply gene amplification using methotrexate and subsequent cloning and banking. GenScript is also fully equipped to execute complete studies on genetic and productive stability in the presence and absence of selective agents. GenScript offers the banking of cell populations as prebanks, masterbanks, and working cell banks.

III. Transient gene expression (TGE)

Stable gene expression is achieved through the insertion of recombinant gene(s) into the host genome. However, the identification and characterization of recombinant cell lines is a costly and time-consuming process. Faster approaches to r-protein production are sometimes preferred, particularly for subcontracting projects or the optimization of large-scale production lines. Rapid protein production (from milligram to gram) using TGE from mammalian cells is well suited to this need. In contrast to stable gene expression, TGE involves short-term r-protein production, typically for not more than10 days post-transfection, in the absence of genetic selection of the plasmid DNA. Our transient gene expression uses suspension-adapted cells and can easily produce as little as a few milliliters or as much as 100 liters. The products, which consist of harvested cell culture fluids or recovered proteins, can be delivered to the client four weeks after an appropriate expression vector has been constructed. Upon request, GenScript can clone the client's gene constructs into high-efficiency vectors designed specifically for the TGE system.

IV. Process development and scale-up

GenScript can optimize the system's growth and production media, also providing a thorough analysis of the growth rate, subcultivation ratio, cell mass, and specific and volumetric productivity using productivity-enhancing conditions. Also, a scale-down model can be applied to predict cell performance. GenScript employs a novel and highly productive proprietary system which is featured with predictive high-throughput.
¹⁵N- or ¹³C-labeled protein expression.

V. Purification

After the expression time has elapsed, the cultures are harvested and the supernatant or cell fraction is collected from the facility for protein purification. Protein purification is typically conducted by affinity column, but other columns, including gel filtration, ion exchange and hydrophobic, are available as well. The purification tag is removed via a specific protease, and the protein is further purified by gel-filtration.
¹⁵N- or ¹³C-labeled protein expression.

VI. Protein characterization

Upon completion of each project, our specialists confirm that the final product meets the client's exact specifications. Typically, this assessment includes any or all of the following: SDS or native PAGE followed by appropriate staining, protein concentration determinations, western blotting, absorption spectrum, N-terminal sequencing, mass spectrometry, NMR spectroscopy, and activity assays.

Service Specifications:

Services	Description	Deliverables
I. Generation of recombinant vector construct	Amplification/isolation of the gene or synthetic gene of interest into a mammalian cell expression vector Verification of the subcloned gene by qPCR and sequencing	Expression vector with insert (5 ug) Verified sequence file Glycerol stock with vector Documentation of procedures
II.Transient protein expression	Transfection of target cells (293, CHO) with recombinant construct DNA Transient protein expression Testing for expression of the recombinant protein by western blot Establishment of stable cell line	Expression data Transient or stable protein expression cell lines Conditioned media
III. Small-scale protein expression	Pilot expression analysis and optimization Generation of cell bank Small-scale expression from one to five liters	Cell pellet Expression data
IV. Large-scale protein production	Cell harvest Testing of expression by western blot Large-scale production up to 100 L	Cell pellet Expression data
V. Protein purification and characterization	Protein purification by affinity column, ion exchange, gel filtration, or hydrophobic column Removal of tags Characterization by western blot, SDS-PAGE, or sequencing	Purified proteins QC data Documentation of procedures

Quotations and Ordering:

To request a quotation, please download and complete the Quotation/Order Form, and send it to us by email or fax.

To order, please download and complete the Quotation/Order Form and send it to us by email or fax with a formal PO (Purchase Order) or credit card information. You can also submit PO/credit card information by phone or via our secure web server.

If submitting samples, please mail them together with a hard copy of the completed Quotation/Order Form to: Protein Services, GenScript, 120 Centennial Ave., Suite 105, Piscataway, NJ 08854, US.

For questions, or to inquire about the status of your order contact us by email, phone, fax or via our secure web server.

Our customer service representatives are available 24 hours a day, Monday through Friday to assist you.

	Email: protein@genscript.com
	Phone: 1-877-436-7274 (Toll-Free) 1-732-885-9188
	Fax: 1-732-210-0262 1-732-885-5878