Overproduction of a human snRNP-associated Sm-D autoantigen in Escherichia coli and Saccharomyces cerevisiae.
To conduct functional and autoimmunity studies, we overproduced human Sm-D1 (hSm-D1), a small nuclear ribonucleoprotein 'core' protein and autoantigen, in Escherichia coli and Saccharomyces cerevisiae. Optimal expression in these organisms was achieved by designing vectors that synthesized abundant hSm-D1 mRNA under the control of the strong, regulatable promoters: T7 phi 10 (E. coli) and GAL1 (yeast). In addition, efficient translation initiation of the hSm-D1 coding sequence was effected in E. coli by utilizing a two-cistron approach; for expression in yeast, we created a 5' untranslated leader whose sequence was based on the consensus of highly expressed genes in S. cerevisiae. The hSm-D1 protein accumulated at high levels in both bacteria and yeast, representing, respectively, approx. 10% and 7% of the total protein. However, in comparison with the authentic protein, the recombinant hSm-D1 displayed different immunoreactive determinants as assessed by Western blot. We thus conclude that certain hSm-D1 immunologic properties are most likely dependent on posttranslational modifications that take place in the cells of higher eukaryotes.
Rokeach
L A
LA
Agouron Institute, La Jolla, CA 92037.
Haselby
J A
JA
Hoch
S O
SO
D001324:Autoantigens / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics* / Q000276:immunology; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D055517:snRNP Core Proteins
D001324:Autoantigens; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins