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

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TypeArticle
Journal titleJournal Of Biological Chemistry
Volume266
Issue5
Pages28312835; # of pages: 5
AbstractSpecific 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.
Publication date
Linkhttp://www.jbc.org/content/266/5/2831.abstract
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedYes
NRC numberSUNG1991
NPARC number9367520
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Record identifierb1286474-7a63-41a6-a32b-cf21067db1c8
Record created2009-07-10
Record modified2016-05-09
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