Gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri: cloning and expression in Escherichia coli

Abstract

A library of cloned Spiroplasma citri genomic sequences was constructed by incorporating HindIII digestion fragments into the plasmid vector pBR328. Immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of S. citri. The spiralin produced by the Escherichia coli transformant was characterized by immunological detection with monoclonal antibody after Western blotting of two-dimensional (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide) electrophoresis gels and by partial proteolytic mapping. The gene for spiralin occurred within a 6.5-kilobase-pair cloned DNA fragment. Spiralin in E. coli was produced regardless of the orientation of the insert within the pBR328 vector. A spiroplasmal DNA sequence which acted as a promoter in E. coli was cloned along with the structural spiralin gene which is expressed in E. coli from that sequence.