Efficient production of biologically active human prolactin in Escherichia coli.
To obtain an adequate amount of human prolactin (hPRL) for elucidation of the structure-function relationship, we have expressed the hPRL cDNA in Escherichia coli (E. coli) by using a high-expression vector. The vector contained a chimeric gene encoding a fusion of protein A, a peptide sensitive to collagenase digestion and hPRL, which was inserted downstream of the right direction promotor of lambda phage. The resulting protein fusion was purified through three column chromatographies of immunoglobulin G-linked Sepharose 4B, DEAE-5PW, and phenyl-5PW. In a typical experiment, a final sample with a purity of more than 80% was obtained with a recovery of more than 40% judged by enzyme-linked immunosorbent assay (ELISA). The fusion thus obtained was digested with collagenase, and protein reactive to anti-hPRL antibody was purified through phenyl-5PW column chromatography. The hPRL sample was found to be identical to authentic hPRL with respect to the amino acid composition and an N-terminal sequence of 20 residues, except that it contained an additional four amino acids at the N-terminal end. This peptide was presumed to be derived from the collagenase-target sequence. The hPRL thus obtained was found to be as active as the authentic hormone either immunologically judged by ELISA or biologically judged by the growth stimulatory effect on rat Nb2 lymphoma cells.
Hiraoka
Y
Y
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan.
Nomata
Y
Y
Matsuo
K
K
Tsubota
N
N
Tanabe
K
K
Fukasawa
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D001681:Biological Assay; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D003012:Microbial Collagenase / Q000378:metabolism; D008969:Molecular Sequence Data; D011388:Prolactin / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000596:Amino Acids; D011993:Recombinant Fusion Proteins; D011388:Prolactin; D003012:Microbial Collagenase