Expression and purification of recombinant active prostate-specific antigen from Escherichia coli.
Human prostate-specific antigen (PSA), a 33 kDa serine protease with comprehensive homology to glandular kallikrein, is secreted from prostatic tissue into the seminal fluid and enters into the circulation. The level of PSA increases in the serum of patients with prostatic cancer and hence is widely employed as a marker of the disease status. In particular, an enzymatically active PSA that is a form cleaved at the N-terminal seven-amino-acids prosequence, APLILSR, of proPSA may play an important roll in the progression of prostate cancer. Thus, the presence of the active form would selectively discriminate the cancer from benign prostatic hyperplasia. In this study, we developed a convenient purification method for the acquisition of active PSA and proPSA. Recombinant proPSA and active PSA were expressed directly in Escherichia coli, easily and efficiently isolated from inclusion bodies, refolded, and purified. Moreover, the enzymatic activity of the recombinant active PSA was confirmed as serine protease using chromogenic chymotrypsin substrate. This purified active PSA could be further applied to scrutinize the biological or conformational characteristics of the protein and to develop specific diagnostic and/or therapeutic agents against prostate cancer.
Jeong
Sujin
S
Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul 140-714, Korea.
Lee
Seong-Wook
SW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002863:Chromogenic Compounds / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D002479:Inclusion Bodies / Q000737:chemistry; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002863:Chromogenic Compounds; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D017430:Prostate-Specific Antigen