High-level secretion of biologically active recombinant human macrophage inflammatory protein-1alpha by the methylotrophic yeast Pichia pastoris.
The human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) was produced at a high level in Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 promoter. To ensure proper folding and secretion of the recombinant polypeptide, the MIP-1alpha gene had been fused to the Saccharomyces cerevisiae alpha-factor prepropeptide. As was revealed by analysis of the cell culture supernatant of recombinant Pichia pastoris, MIP-1alpha was efficiently secreted. Immunoblot analysis of secreted proteins from recombinant clones using a polyclonal antibody directed against MIP-1alpha revealed an apparent molecular mass of 8 kDa for the recombinant polypeptide. Up to 70 mg of MIP-1alpha was purified from 1 liter of yeast culture supernatant by a single chromatography step. Biological activity of recombinant MIP-1alpha was shown in a chemotaxis assay. Here, the polypeptide specifically induced migration of U937 cells expressing the CCR1 (MIP-1alpha receptor). Also, in competition binding assays the recombinant MIP-1alpha displayed high affinity binding.
Maeda
Y
Y
Abteilung Molekulare Membranbiologie, Max-Planck-Institut für Biophysik, Heinrich-Hoffmann-Strasse 7, Frankfurt/M., D-60528, Germany. maeda@mpibp-frankfurt.mpg.de
Kuroki
R
R
Suzuki
H
H
Reiländer
H
H
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D002633:Chemotaxis / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D017931:DNA Primers; D004274:DNA, Recombinant; D019402:Macrophage Inflammatory Proteins; D011994:Recombinant Proteins