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Production of recombinant human alpha(1)-microglobulin and mutant forms involved in chromophore formation

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Author

Summary, in English

alpha-Microglobulin, a 26 kDa lipocalin present in plasma and tissues, carries a set of unknown chromophores, bound to C34, K92, KI 18 and KI 30, which cause its charge and size heterogeneity. In man, the protein is found in two forms, full length and lacking the C-terminal tetrapeptide LIPR (t-alpha(1)-microglobulin), both which are heme-binding and the latter with heme-degrading properties. We report cloning and overexpression of full length alpha(1)-microglobulin (wt protein), t-alpha(1)-microglobulin (wtz LIPR) and the mutants C34S, K(92,118,130)T and C34S/K(92,118,130)T, the latter subsequently abbreviated as K(3)T and C34S/K(3)T, in Escherichia coli. After purification and refolding from inclusion bodies, all proteins were correctly folded as determined by far-UV circular dichroism and radioimmunoassay. As revealed by gel filtration, recombinant alpha(1)-microglobulins had lower tendencies to form dimers than human plasma or urine analogues. All alpha(1)-microglobulin forms displayed higher amounts of the chromophore than bovine serum albumin but significantly lower than the human urine or plasma counterparts. Differences in the absorbance and fluorescence profiles are consistent with a model where the chromophore is formed by a series of reactions with heme or other chromophore precursors and where C34 is essential for binding of the ligand, K92, KI 18 and K130 are involved in transformation into the chromophore and LIPR inhibits the latter reaction. (c) 2006 Elsevier Inc. All rights reserved.

Publishing year

2007

Language

English

Pages

145-152

Publication/Series

Protein Expression and Purification

Volume

53

Issue

1

Document type

Journal article

Publisher

Academic Press

Topic

  • Infectious Medicine
  • Endocrinology and Diabetes

Keywords

  • affinity chromatography
  • immobilised metal-ion
  • alpha 1-microglobulin
  • recombinant protein
  • circular dichroism
  • radioimmunoassay

Status

Published

Research group

  • Molecular Endocrinology

ISBN/ISSN/Other

  • ISSN: 1046-5928