Keratinocyte growth factor-2 production in an ompT-deficient Escherichia coli K-12 mutant.
A variant form of Keratinocyte growth factor-2 (KGF-2) spanning amino acids A63-S208 was produced in the Escherichia coli K-12 host W3110. When the protein was purified using a standard process, the first six N-terminal amino acids were rapidly and specifically removed from the protein. This cleavage resulted in a truncated KGF-2 species (S69-S208). To circumvent this problem, guanidine-HCl was used to inhibit the putative proteolytic activity. This modified process resulted in a massive loss of protein product due to precipitation, in addition to the cost and corrosiveness of guanidine-HCl. To develop an economically feasible, scaleable, and robust process for KGF-2 production, we were tasked with identifying the protease(s) responsible for the N-terminal degradation. Experimental evidence revealed that OmpT (outer membrane protein T) was the primary protease involved in the N-terminal cleavage of the A63-S208 KGF-2 protein. Moreover, the OmpT-mediated cleavage occurred at a novel site (Arg-Ser). From this work, we show that production of the A63-S208 form of KGF-2 in an ompT-deleted E. coli host nearly abolished the N-terminal cleavage issue.
Laird
Michael W
MW
Fermentation & Cell Culture Sciences, Human Genome Sciences, Inc., Rockville, Maryland 20850, USA. mike_laird@hgsi.com
Cope
Karen
K
Atkinson
Ronald
R
Donahoe
Mark
M
Johnson
Kelly
K
Melick
Meredith
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005240:Feasibility Studies; D051526:Fibroblast Growth Factor 10; D005346:Fibroblast Growth Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D051526:Fibroblast Growth Factor 10; D005346:Fibroblast Growth Factors; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease