Expression in Escherichia coli of the genes coding for reaction center subunits from Rhodobacter sphaeroides: wild-type proteins and fusion proteins containing one or four truncated domains from Staphylococcus aureus protein A at the carboxy-terminus.
Gene cassettes were constructed containing Rhodobacter sphaeroides puhA, pufM and pufL sequences with synthetic 5' ends for production in Escherichia coli of the H, M and L subunits of the photosynthetic reaction center. In addition, gene cassettes coding for fusion proteins with proteinase recognition site(s) between the amino-terminal part of H, M or L subunits, and the carboxy-terminal part consisting of one (B') or four (D'ABC') domains of Staphylococcus aureus protein A were constructed. A modified expression vector pDS12/RBSII containing the T5 promoter PN25, the lac operator, and a newly inserted E. coli lipoprotein ribosome-binding site was used. Inducible synthesis of plasmid-encoded polypeptides was accompanied by reduced growth. The products comigrated with R. sphaeroides reaction center subunits H, M and L. They were identified by Western blot experiments using antibodies raised against reaction center proteins. The hybrid protein containing the reaction center H subunit fused to the single domain B' was not detected by nonspecific antisera. In contrast, the three fusion proteins containing domains D'ABC' were identified using nonspecific antisera. This indicated that domains D'ABC' were sufficient to bind to the Fc part of IgG molecules, whereas domain B' was not sufficient. This property was used to purify all three fusion proteins with domains D'ABC' by affinity chromatography from the membrane fraction of E. coli cells.
Söhlemann
P
P
Max-Planck-Institut für Biophysik, Frankfurt/Main, F.R.G.
Oeckl
C
C
Michel
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000235:genetics*; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000302:isolation & purification
D010446:Peptide Fragments; D045322:Photosynthetic Reaction Center Complex Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A