Production and activation of recombinant hK2 with propeptide mutations resulting in high expression levels.
Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 microgram.mL-1), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg.L-1). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild-type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15-40-fold giving 10-30 mg hK2.L-1. These results indicate that the low expression levels of wild-type hK2 are related to the activation or autoactivation of the wild-type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild-type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.
Lövgren
J
J
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden. janita.lovgren@utu.fi
Tian
S
S
Lundwall
A
A
Karp
M
M
Lilja
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008297:Male; D009154:Mutation; D011467:Prostate / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D020840:Tissue Kallikreins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D020840:Tissue Kallikreins