Secretion of active kringle-2-serine protease in Escherichia coli.
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.
Obukowicz
M G
MG
Corporate Research and Development Staff, Monsanto Company, St. Louis, Missouri 63198.
Gustafson
M E
ME
Junger
K D
KD
Leimgruber
R M
RM
Wittwer
A J
AJ
Wun
T C
TC
Warren
T G
TG
Bishop
B F
BF
Mathis
K J
KJ
McPherson
D T
DT
D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D010959:Tissue Plasminogen Activator