Expression of the human complex-forming glycoprotein HC (alpha 1-microglobulin) in Escherichia coli.
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 of human protein HC.
Calero
M
M
Servicio de Endocrinología, Hospital Ramón y Cajal, Madrid, Spain.
Méndez
E
E
García
E
E
D000510:Alpha-Globulins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014357:Trypsin
D000510:Alpha-Globulins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C016001:alpha-1-microglobulin; D014357:Trypsin