title: Production and purification of novel secreted human proteins.
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 biological activity due to these differences have been demonstrated. The BV system has served as a viable alternative for the high throughput, high fidelity expression of many novel secreted human genes. To date, more than 75 new genes have been expressed, and the re-proteins purified. This expression system combines many favorable traits including relative speed, moderate cost but perhaps most importantly, the production of biologically active proteins.
authors.affiliation: Human Genome Sciences Inc., Rockville, MD 20850-3338, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Coleman
authors.forename: D
authors.initials: D
authors.lastname: Parmelee
authors.forename: N R
authors.initials: NR
authors.lastname: Thotakura
authors.forename: N
authors.initials: N
authors.lastname: Nguyen
authors.forename: M
authors.initials: M
authors.lastname: Bürgin
authors.forename: S
authors.initials: S
authors.lastname: Gentz
authors.forename: R
authors.initials: R
authors.lastname: Gentz
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D018925:Chemokines / Q000235:genetics*; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006133:Growth Substances / Q000235:genetics*; D006728:Hormones / Q000235:genetics*; D006801:Humans; D010802:Phylogeny
chemical_list: D018925:Chemokines; D006133:Growth Substances; D006728:Hormones