Production of the immunoglobulin variable domain REIv via a fusion protein synthesized and secreted by Staphylococcus carnosus.
REIv--the variable domain of an immunoglobulin x light chain--was produced by heterologous gene expression in a Gram-positive bacterium, purified to homogeneity and characterized. A host/vector combination based on secretion of Staphylococcus hyicus lipase by Staphylococcus carnosus was exploited. A gene encoding a fusion protein, composed of an aminoterminal portion of the pre-pro-peptide of S. hyicus lipase, a hexahistidine affinity tag, followed by the recognition sequence of IgA protease and REIv was constructed. Expression of the fusion gene in S. carnosus causes selective secretion and accumulation of a soluble fusion protein in the culture medium (5-10 mg/l), which can be purified from the supernatant by immobilized metal ion affinity chromatography (IMAC). REIv is released from the fusion protein with an additional threonine and proline residue at the aminoterminus (REIvTP) by site-specific cleavage with IgA protease and can be separated from the hexahistidine-tagged fusion partner and the protease by a second passage through an IMAC gel matrix. Like authentic REIv, the isolated protein (> 1 mg/l culture medium) migrates as a dimer in gel filtration chromatography and undergoes cooperative, reversible unfolding in urea. The isolated immunoglobulin REIvTP and authentic REIv have indistinguishable free energies of unfolding (approx. 26 kJ/mol, 6.3 kcal/mol).
Pschorr
J
J
Institut für Molekulare Genetik, Georg-August-Universität Göttingen, Germany.
Bieseler
B
B
Fritz
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001536:Bence Jones Protein / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000502:physiology; D006868:Hydrolysis; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013210:Staphylococcus / Q000378:metabolism*; D014508:Urea
D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins; D014508:Urea; D001536:Bence Jones Protein