Production of recombinant human alpha1-microglobulin and mutant forms involved in chromophore formation.
Alpha(1)-Microglobulin, a 26 kDa lipocalin present in plasma and tissues, carries a set of unknown chromophores, bound to C34, K92, K118 and K130, 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 (wtdeltaLIPR) 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, K118 and K130 are involved in transformation into the chromophore and LIPR inhibits the latter reaction.
Kwasek
Anna
A
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakow, Poland. akwasek@mol.uj.edu.pl
Osmark
Peter
P
Allhorn
Maria
M
Lindqvist
Annika
A
Akerström
Bo
B
Wasylewski
Zygmunt
Z
D000510:Alpha-Globulins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000652:urine; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007525:Isoelectric Focusing; D008024:Ligands; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014170:Transformation, Genetic
D000510:Alpha-Globulins; D008024:Ligands; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D006418:Heme