Open AccessDetection and characterization of a mouse alpha-spectrin cDNA clone by its expression in Escherichia coli.
Author(s) -
Livia Cioè,
Peter J. Curtis
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.5.1367
Subject(s) - spectrin , complementary dna , microbiology and biotechnology , escherichia coli , biology , antiserum , clone (java method) , expression vector , antibody , gene , biochemistry , genetics , cell , recombinant dna , cytoskeleton
A cloned segment of mouse alpha-spectrin mRNA has been identified by immunological techniques. Double-stranded cDNA derived from spleens of anemic mice was introduced into a bacterial expression vector, pUC, and transformed Escherichia coli colonies were screened by using an antiserum to erythrocyte membrane ghost proteins. Of 17 positive colonies, 2 bound antibody to mouse spectrin, and these 2 colonies contained 750-base-pair inserts that cross-hybridized. Transfer of the 750-base-pair insert to an expression vector containing the PL promoter of phage lambda produced larger amounts of peptides that were bound by antibody to mouse spectrin. The spectrin-like peptides made in E. coli elicited antibody that reacted only with the alpha-spectrin subunit of erythrocyte membranes. This clone will be useful for the study of the structure and expression of the spectrin gene, particularly in understanding the role of spectrin in human inherited hemolytic anemias.