Production and purification of novel secreted human proteins

Gene
T A ColemanR Gentz

Abstract

Our objective during the last year was to produce and purify 50-80 novel, secreted human proteins identified via high throughput cDNA sequencing and computer analysis. We chose the baculovirus expression vector system in order to obtain secreted, correctly folded, bioactive proteins. Recombinant (re-)baculoviruses (BV) were plaque purified, and pulse-labeling was used to verify the synthesis and secretion of the re-proteins. N-terminal microsequencing was performed to simultaneously confirm the identity of the protein(s) as well as the signal peptide (SP) cleavage site(s). Following sequence confirmation, the proteins were purified to homogeneity and functional assays carried out to determine potential therapeutic applications. We identified proteins with antiviral activity, several novel growth factors, proteins influencing the differentiation of specific cell types, novel proteases and protease inhibitors among others. Certain proteins were expressed both in insect cells and in CHO stable cell lines. In the cases analyzed, we found that the same SP cleavage site was utilized in the two expression systems. Significant differences were observed in the carbohydrate moieties attached to the proteins, though no effects on the biol...Continue Reading

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Citations

Jun 27, 2002·Biochimica Et Biophysica Acta·Seyung ChungBrian C Rymond
Jan 28, 2003·Drug Discovery Today·Stephen P Chambers
Feb 6, 2002·Current Opinion in Chemical Biology·Michele Gilbert, Joanna S Albala
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Feb 12, 1998·The American Journal of Physiology·K L MadsenR N Fedorak
Feb 7, 2001·The Journal of Immunology : Official Journal of the American Association of Immunologists·K TamadaL Chen

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