Secretion of authentic 20-kDa human growth hormone (20K hGH) in Escherichia coli and properties of the purified product.
Using Bacillus amyloliquefaciens neutral protease gene (npr), we have constructed a secretion system of 20-kDA human growth hormone (20K hGH) in E. coli. The secretion-signal region from npr was modified inserting a fragment coding a 2Lys-5Leu cluster. In this system we found that co-expression of glutathione reductase remarkably increased accumulation level of 20K hGH in periplasm and confirmed that secreted 20K hGH was correctly processed. The recombinant 20K hGH was highly purified and subjected to analyses of physicochemical properties and biological activities which are still unclear and controversial due to difficulty in preparing the sample with authentic structure. The secreted recombinant product had authentic disulfide linkages and showed molecular weight of 20,270.5 +/- 3.7 (theoretical value, 20,269.9). The results suggest that the recombinant 20K hGH is a full agonist on rat growth promotion and lipolysis stimulation in isolated rat adipose tissues. In particular, the lipolysis-stimulating activity of 20K hGH was distinct as compared with that of 22K hGH under physiological concentration. Cell proliferation activity via prolactin-receptor in Nb-2 lymphoma was obviously low as compared with that of 22K hGH.
Uchida
H
H
Life Science Laboratories, Mitsui Toatsu Chemicals Inc., Chiba, Japan.
Naito
N
N
Asada
N
N
Wada
M
M
Ikeda
M
M
Kobayashi
H
H
Asanagi
M
M
Mori
K
K
Fujita
Y
Y
Konda
K
K
Kusuhara
N
N
Kamioka
T
T
Nakashima
K
K
Honjo
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D019382:Human Growth Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D019382:Human Growth Hormone