Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli.
The hGH-V (or hGH-2) gene codes for human placental growth hormone (hPGH). Secretion of hPGH is continuous, in contrast with the pulsed secretion of pituitary growth hormone (hGH) which it progressively replaces in the maternal bloodstream. hGH-V cDNA has previously been cloned and isolated. Analysis of its nucleotide sequence has revealed a 191-residue protein, hPGH, differing from hGH at 13 positions. The calculated pI is more basic than that of the pituitary hormone. Here we have inserted hGH-V cDNA into the pIN-III-ompA3 plasmid in order to produce hPGH in its native form in Escherichia coli D1210. Expression of hGH-V cDNA in E. coli is significantly lower than that of hGH cDNA with the same expression system. The hPGH produced in E. coli was purified in quantities sufficient to allow its biochemical and immunochemical characterization. The molecular mass of the protein was determined by electrospray m.s. The determined mass, 22,320 Da, agrees well with the molecular mass calculated from the translated cDNA sequence, assuming the presence of two disulphide bridges. Having established the technique for producing hPGH with a primary structure identical to the natural, non-glycosylated, 22 kDa isoform, we can now plan the full physicochemical and pharmaceutical characterization of this new hormonal entity.
Igout
A
A
Biochimie et Laboratoire d'Endocrinologie, Tour de Pathologie, Liege, Belgium.
Van Beeumen
J
J
Frankenne
F
F
Scippo
M L
ML
Devreese
B
B
Hennen
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010926:Placental Hormones / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D018076:DNA, Complementary; C064980:GH2 protein, human; D010926:Placental Hormones; D011994:Recombinant Proteins; D013006:Growth Hormone