Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli

Shizhe Jia, Robert P. Mee, Gerald Morford, Neera Agrwal, Patricia G. Voss, Ioannis K. Moutsatsos, John L. Wang

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a λgt11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing β-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by affinity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against β-galactosidase. These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli. Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.

Original languageEnglish (US)
Pages (from-to)197-204
Number of pages8
JournalGene
Volume60
Issue number2-3
DOIs
StatePublished - 1987

Keywords

  • Northern blotting
  • Recombinant DNA
  • cDNA clone
  • fusion protein
  • immunoblot screening
  • lysogen
  • peptide mapping
  • phage λgt vector

ASJC Scopus subject areas

  • Genetics

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