Specific degenerate codons enhanced selective expression of human parathyroid hormone in Escherichia coli.
Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli. With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter. In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84). Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l). With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E. coli-favored codon degeneracy. Expression yielded the intact PTH at 20 mg/liter. Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample. Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.
Sung
W L
WL
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Zahab
D M
DM
Barbier
J R
JR
Watson
D
D
Yaguchi
M
M
Neugebauer
W
W
Willick
G E
GE
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics*; D010957:Plasmids
D003062:Codon; D010281:Parathyroid Hormone