Human parathyroid hormone: efficient synthesis in Escherichia coli using a synthetic gene, purification and characterization.
Human parathyroid hormone is a peptide hormone consisting of 84 amino acid residues. Production of small proteins by direct expression in Escherichia coli is often unsuccessful owing to susceptibility of the mRNA and/or the product to endogenous enzymes. In this study, direct expression of the hormone at an excellent level (over 100 mg/L) has been achieved by using a suitably designed synthetic gene under the control of the T7 promoter. The protein produced in bacteria was extracted and easily purified in a good yield of 27 mg/L. The purified product was physico-chemically identified as intact human parathyroid hormone from the results of amino acid analysis, N-terminal sequencing, and peptide mapping using fast atom bombardment mass spectrometry. In biological assays the purified product stimulated adenylate cyclase in vitro, promoted bone growth and increased the serum osteocalcin in rats to the same extent as the authentic hormone.
Oshika
Y
Y
Chemistry Research Laboratory, Takeda Chemical Industries, Ltd., Osaka, Japan.
Yamada
T
T
Nakagawa
S
S
Fujishima
A
A
Kawase
M
M
Ishibashi
Y
Y
Fukuda
T
T
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003062:Codon; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics
D003062:Codon; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D004247:DNA