Elsevier

Analytical Biochemistry

Volume 122, Issue 2, 15 May 1982, Pages 348-359
Analytical Biochemistry

Characterization of intact and trypsin-digested biosynthetic human growth hormone by high-pressure liquid chromatography

https://doi.org/10.1016/0003-2697(82)90294-9Get rights and content

Abstract

The structural properties of purified human growth hormone (hGH) produced by Escherichia coli K-12 into which the hGH gene has been inserted have been fully characterized by high-pressure liquid chromatography of native hGH and tryptic digests of hGH. All of the tryptic peptides have been separated by high-pressure liquid chromatography and their sequence determined. Comparison of the primary structure with that of the purified pituitary-derived hGH has established the integrity of the biosynthetic hGH disulfide arrangement and amino acid sequence with the presence of an extra NH2-terminal methionine.

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Cited by (57)

  • The proteomics of N-terminal methionine cleavage

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    Citation Excerpt :

    If the protein has an N terminus associated with a risk of non-processing, various methods may be used to prevent Met retention: (i) adjusting expression conditions to optimize MAP activity according to the rate of protein synthesis in vivo (58), (ii) in vitro processing with purified wild-type or engineered MAP (59–62), (iii) dual co-overproduction of MAP and the protein of interest in vivo (25, 63, 64), (iv) processing in vitro or in vivo of the N terminus with another amino- or endoprotease (65–67), or (v) fusion of the ORF to a propeptide or ubiquitin sequence and use of endogenous signal peptidase specificity (68, 69). This last strategy has also proved successful for residues that cannot be unmasked by NME (this study, Fig. 3), such as the thrombin inhibitor hirudin, which must have Ile at its N terminus for biological activity, or human growth hormone, which must have Phe at its N terminus (70–73). In Archaea, most mature proteins have Ala, Met, Gly, Ser, Thr, or Pro at their N terminus (10).

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An abstract of these results was presented at the International Symposium on High-Pressure Liquid Chromatography of Proteins and Peptides, held in Washington, D. C., on November 16–17, 1981.

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