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Secretion of active kringle-2-serine protease in Escherichia coli
M G Obukowicz1, M E Gustafson, K D Junger
1Corporate Research and Development Staff, Monsanto Company, St. Louis, Missouri 63198.
Abstract:
Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.
Insights
Recombinant human tissue plasminogen activator variant (MB1004) produced in E. coli shows higher activity than glycosylated mammalian variants. This highlights the impact of glycosylation on assessing enzyme specific activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Active human tissue plasminogen activator variant (MB1004) comprises Kringle-2 and serine protease domains.
- MB1004 is a complex, 38 kDa protein with nine disulfide bonds, requiring efficient production methods.
Purpose of the Study:
- To synthesize and characterize a secreted, active form of MB1004 in Escherichia coli.
- To compare the specific activity of E. coli-produced MB1004 with glycosylated and deglycosylated mammalian-produced variants.
Main Methods:
- MB1004 was secreted into E. coli periplasm using PhoA leader peptide and tac promoter.
- Purification involved lysine and Erythrina inhibitor affinity chromatography.
- Specific activity was assessed using four in vitro assays, comparing E. coli MB1004, glycosylated MB1004G, and deglycosylated MB1004N.
Main Results:
- Soluble, active MB1004 was purified from E. coli homogenates.
- E. coli-derived MB1004 and deglycosylated MB1004N exhibited similar specific activities.
- Both MB1004 and MB1004N showed 4-12 fold higher specific activity than glycosylated MB1004G.
Conclusions:
- Glycosylation significantly affects the measured specific activity of MB1004.
- E. coli secretion provides a method for producing active, non-glycosylated MB1004 suitable for structure-function studies.
- Accurate assessment of enzyme activity requires consideration of post-translational modifications like glycosylation.