In Vitro Deletional Mutagenesis for Bacterial Production of the 20,000-Dalton Form of Human Pituitary Growth Hormone

John P. Adelman, Joel S. Hayflick, Mark Vasser, Peter H. Seeburg

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

The 20,000-dalton (20K) variant form of human growth hormone (hGH) present in extracts from pituitary glands differs from the major form of hGH (22K, 191 amino acids) by the deletion of amino acid residues 32–46. Using oligonucleotide-mediated mutagenesis, the DNA coding for these amino acids was deleted from the gene previously constructed by us (Goeddel et al., 1979) for microbial hGH production. The DNA to be deleted was looped out by the annealing of a synthetic oligodeoxyribonucleotide to the coding strand of the hGH gene contained on recombinant phage M13 mp8 DNA. Resulting heteroduplex structures were stabilized using primer-directed in vitro DNA synthesis in the presence of T4 DNA ligase. On transformation of Escherichia coli, these heteroduplex DNAs yielded phage whose genomes contained either the original or the partially deleted hGH gene, and genotypes were distinguished by in situ plaque hybridization with synthetic oligonucleotide probes. A gene with the correct deletion was used to express the short hGH variant in E. coli.

Original languageEnglish (US)
Pages (from-to)183-193
Number of pages11
JournalDNA
Volume2
Issue number3
DOIs
StatePublished - Sep 1983

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'In Vitro Deletional Mutagenesis for Bacterial Production of the 20,000-Dalton Form of Human Pituitary Growth Hormone'. Together they form a unique fingerprint.

  • Cite this