PMID: 7539295May 18, 1995Paper

Expression of the human complex-forming glycoprotein HC (alpha 1-microglobulin) in Escherichia coli

Biochimica Et Biophysica Acta
M CaleroE García

Abstract

The mature form of human protein HC, or alpha 1-microglobulin, has been expressed in Escherichia coli. Protein HC is a member of the lipocalin superfamily of hydrophobic ligand-binding proteins, and carries a heterogeneous chromophore linked covalently by a reduction-resistant bond. Protein HC was first overexpressed as a C-LytA/HC fusion protein containing the C-terminal moiety of the pneumococcal lytic amidase (LytA). Recombinant C-LytA/HC was found to be an insoluble aggregate that was solubilized with 6 M guanidinium chloride and renatured by the addition of thiol reagents in the presence of L-arginine. Recombinant protein HC (rHC) was released from C-LytA/HC by trypsin digestion and purified by size-exclusion chromatography. rHC protein possesses an N-terminal amino-acid sequence identical to that of human protein HC, and a slightly lower molecular mass as determined by SDS-PAGE. Both C-LytA/HC and rHC reacted with polyclonal antibodies raised against native protein HC. A photodiode array detection system on-line with a HPLC system has allowed the identification of a chromophore associated to rHC protein displaying significant absorption in the visible region of the spectrum in resemblance to that found in the natural form...Continue Reading

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Citations

Dec 16, 2006·Protein Expression and Purification·Anna KwasekZygmunt Wasylewski
Nov 4, 2000·Biochimica Et Biophysica Acta·B AkerströmA Lindqvist
Feb 17, 2000·Journal of Chromatography. B, Biomedical Sciences and Applications·H Husi, M D Walkinshaw

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