Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine-165 to alanine.
We have mutated a synthetic human growth hormone (hGH) gene specifically at the codon for Cys-165 to a codon for Ala by replacement of synthetic deoxyoligonucleotides corresponding to this site. This modification prevented the formation of a disulfide bond between Cys-53 and Cys-165 in the hGH molecule. This mutated protein, [Ala165]hGH was expressed at the same level as the intact hGH, 4 X 10(5) molecules per cell under the control of the tryptophan promoter in Escherichia coli, and retained similar immunological activity to intact hGH. The limited digestion pattern of the mutated protein with human plasmin suggests that the tertiary structure of [Ala165]hGH resembles to that of the intact hGH molecule. [Ala165]hGH revealed full biological activity as examined by the body weight increase of hypophysectomized rats.
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003062:Codon; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005796:Genes; D013006:Growth Hormone / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids
D000596:Amino Acids; D003062:Codon; D009838:Oligodeoxyribonucleotides; C047614:somatotropin, Ala(165)-; D013006:Growth Hormone; D003545:Cysteine; D000409:Alanine