title: Optimization of BLyS production and purification from Escherichia coli.
abstract: B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.
authors.affiliation: Fermentation and Cell Culture Sciences, Human Genome Sciences, Inc., Rockville, MD 20850, USA. mike_laird@hgsi.com
authors.forename: Michael W
authors.initials: MW
authors.lastname: Laird
authors.forename: Gavin C
authors.initials: GC
authors.lastname: Sampey
authors.forename: Kelly
authors.initials: K
authors.lastname: Johnson
authors.forename: David
authors.initials: D
authors.lastname: Zukauskas
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pierre
authors.forename: June S
authors.initials: JS
authors.lastname: Hong
authors.forename: Bridget A
authors.initials: BA
authors.lastname: Cooksey
authors.forename: Yuling
authors.initials: Y
authors.lastname: Li
authors.forename: Olga
authors.initials: O
authors.lastname: Galperina
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Karwoski
authors.forename: Robert N
authors.initials: RN
authors.lastname: Burke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053264:B-Cell Activating Factor; D001402:B-Lymphocytes / Q000502:physiology; D019149:Bioreactors / Q000382:microbiology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005285:Fermentation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D014170:Transformation, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D053264:B-Cell Activating Factor; D003062:Codon; D008565:Membrane Proteins; D011994:Recombinant Proteins; C505720:Tnfsf13b protein, mouse; D014409:Tumor Necrosis Factor-alpha