Specific degenerate codons enhanced selective expression of human parathyroid hormone in Escherichia coli

J Biol Chem. 1991 Feb 15;266(5):2831-5.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Western
  • Chromatography, Ion Exchange
  • Codon
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial
  • Genes, Bacterial
  • Humans
  • Molecular Sequence Data
  • Parathyroid Hormone / genetics*
  • Plasmids

Substances

  • Codon
  • Parathyroid Hormone

Associated data

  • GENBANK/M61891
  • GENBANK/M61892
  • GENBANK/M61893
  • GENBANK/M61894
  • GENBANK/M61895
  • GENBANK/M64270
  • GENBANK/M64271
  • GENBANK/M64485
  • GENBANK/M84326
  • GENBANK/M84327