Proteomics

Recombinant Proteins

CBI is pleased to offer full "paper to protein" cloning as one of our services. This literally means we can start with a DNA or protein sequence as written on a piece of paper and supply you with the corresponding purified recombinant protein. The first step can be de novo synthesis of the gene or PCR amplification of the encoding sequence from genomic or plasmid DNA extracted from the source tissue or cell. This is followed by insertion of the gene into an appropriate expression vector, transfection of the vector into a host cell line, and finally, growth of the cells and purification of the protein. CBI routinely uses bacteria, yeast, and insect cells for overexpression of target proteins.

For proteins destined for compliance submission, CBI strongly recommends a meeting with the appropriate regulatory authorities so that there are no undue surprises down the road. CBI also stresses that commercialization of a recombinant protein will most likely entail licensing issues, which must be settled between the license holders and our potential clients. Our usual path of production of a particular recombinant protein is to perform all the necessary molecular biology so as to create an expression clone, in any suitable host cell, including bacteria, mammalian cells, yeast, or insect cells. Our clients usually provide us with encoding DNA, and CBI then creates and confirms the expression vector.

Once we have an expression vector, CBI recommends that expression of the target protein first be determined in small scale fermentation or pilot scale cell culture. This run would be used to assess expression levels and to determine the best methods for extraction of the protein from the cell paste. Once expression levels are determined, the next steps are to assess scalability of production by doing increasingly larger fermentation runs.

The presence of the target protein can be assessed by any one of several means, including SDS PAGE, mass spectrometry, ELISA, western blot, etc. Purification schemes include lysis of the cells, an initial precipitation step, metal chelate chromatography, one polishing chromatography step. Characterization is usually done by SDS-PAGE, N- terminal sequence analysis, mass spectrometry, ELISA, western blot analysis, etc.



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Commonwealth Biotechnologies, Inc. 601 Biotech Drive, Richmond, VA 23235 800-735-9224 (800-RELY-CBI) FAX: 804-648-2641 email: [email protected]