Cloning and expression of Acholeplasma laidlawii membrane acyl proteins in Escherichia coli.
Many integral membrane proteins in Acholeplasma laidlawii are enriched in hydrophilic amino acid residues and covalently modified with fatty acids. In order to understand how these proteins are inserted and anchored in the bilayer, we have cloned several of the major A. laidlawii proteins in Escherichia coli: 900 recombinant clones containing 4-kbp DNA fragments, inserted into the BamHI site of the plasmid pAT 153, were screened with antibodies. With antimembrane antibodies, 26 positive clones were detected, and with a mixture of five different monospecific antibodies, another 7 clones were obtained. Immunological analysis of the colonies in situ verified that antigens for A. laidlawii membrane proteins D12, T2, T3, T4a, and unidentified proteins were produced in separate clones. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting showed several fragments [25 to 94 kilodalton (kDa)] for each of these proteins, some of which were even visible on Coomassie Blue-stained gels. It is concluded that A. laidlawii membrane proteins can be efficiently expressed in E. coli.
Tegman
V
V
Department of Biochemistry, University of Umeå, Sweden.
Wallbrandt
P
P
Nyström
S
S
Johansson
K E
KE
Jonsson
B H
BH
Wieslander
A
A
D000128:Acholeplasma laidlawii / Q000235:genetics* / Q000276:immunology; D000215:Acylation; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins