Journal Article

Enhanced stability of recombinant keratinocyte growth factor by mutagenesis

Eric Hsu, Timothy Osslund, Rebecca Nybo, Bao-Lu Chen, William C. Kenney, C.Fred Morris, Tsutomu Arakawa and Linda O. Narhi

in Protein Engineering, Design and Selection

Volume 19, issue 4, pages 147-153
Published in print April 2006 | ISSN: 1741-0126
Published online February 2006 | e-ISSN: 1741-0134 | DOI: http://dx.doi.org/10.1093/protein/gzj013
Enhanced stability of recombinant keratinocyte growth factor by mutagenesis

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Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.

Keywords: KGF; protein stability; heparin binding; cysteine replacement

Journal Article.  4715 words.  Illustrated.

Subjects: Proteins

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