Overexpression of native human beta 2-microglobulin in Escherichia coli and its purification.
beta 2-Microglobulin (beta 2M), the small subunit of human leukocyte antigen (HLA) class-I proteins, has been synthesized in Escherichia coli and purified in mg amounts. A beta 2m cDNA clone was fused in-frame behind DNA encoding the signal sequence for the outer membrane protein, OmpA. Three different constructions were made, whose products differed by the insertion of either an extra Ala residue, the hexapeptide AEFLEA [single-letter amino acid (aa) code], or no aa between the OmpA signal sequence and beta 2M-coding sequence. All three protein products were correctly processed by bacterial signal peptidase, as determined by N-terminal sequencing, and all three were secreted as soluble proteins into the periplasmic space. However, the signal sequence of the preprotein with the inserted hexapeptide, AEFLEA, was cleaved to a much greater degree than the other two preproteins. When there was no insertion, the mature protein was identical to human beta 2M, as analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, circular dichroism, and native isoelectric focusing. This 'bacterial beta 2M', radiolabeled with Bolton-Hunter reagent, was able to exchange into papain-solubilized HLA-B7, as determined by Sephadex G-75 chromatography and immune precipitation, indicating that bacterial beta 2M could complex with the heavy chain of HLA-B7.
Parker
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KC
Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138.
Wiley
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DC
D015153:Blotting, Western; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D012697:Serine Endopeptidases; D001613:beta 2-Microglobulin / Q000096:biosynthesis / Q000235:genetics*
D008565:Membrane Proteins; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase