Advanced

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

Kwasek, Anna; Osmark, Peter LU ; Allhorn, Maria LU ; Lindqvist, Annika; Åkerström, Bo LU and Wasylewski, Zygmunt (2007) In Protein Expression and Purification 53(1). p.145-152
Abstract
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
affinity chromatography, immobilised metal-ion, alpha 1-microglobulin, recombinant protein, circular dichroism, radioimmunoassay
in
Protein Expression and Purification
volume
53
issue
1
pages
145 - 152
publisher
Academic Press
external identifiers
  • wos:000246245000018
  • scopus:33847148408
ISSN
1046-5928
DOI
10.1016/j.pep.2006.10.023
language
English
LU publication?
yes
id
3c091488-3275-466f-baab-4a8c574fd64e (old id 661080)
date added to LUP
2007-12-13 18:14:30
date last changed
2017-06-25 03:39:12
@article{3c091488-3275-466f-baab-4a8c574fd64e,
  abstract     = {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.},
  author       = {Kwasek, Anna and Osmark, Peter and Allhorn, Maria and Lindqvist, Annika and Åkerström, Bo and Wasylewski, Zygmunt},
  issn         = {1046-5928},
  keyword      = {affinity chromatography,immobilised metal-ion,alpha 1-microglobulin,recombinant protein,circular dichroism,radioimmunoassay},
  language     = {eng},
  number       = {1},
  pages        = {145--152},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Production of recombinant human alpha(1)-microglobulin and mutant forms involved in chromophore formation},
  url          = {http://dx.doi.org/10.1016/j.pep.2006.10.023},
  volume       = {53},
  year         = {2007},
}