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 journalArticle

15 Citations (Scopus)

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
Externally publishedYes

Fingerprint

Galectin 3
Molecular Cloning
Lectins
Escherichia coli
Peptides
Galactosidases
Complementary DNA
Clone Cells
Galactose
Proteolysis
3T3 Cells
Peptide Mapping
Antibody Affinity
Affinity Chromatography
Immunoblotting
Sodium Dodecyl Sulfate
Northern Blotting
Sepharose
Polyacrylamide Gel Electrophoresis
Intercellular Signaling Peptides and Proteins

Keywords

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

ASJC Scopus subject areas

  • Genetics

Cite this

Jia, S., Mee, R. P., Morford, G., Agrwal, N., Voss, P. G., Moutsatsos, I. K., & Wang, J. L. (1987). Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli. Gene, 60(2-3), 197-204. https://doi.org/10.1016/0378-1119(87)90228-9

Carbohydrate-binding protein 35 : molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli. / Jia, Shizhe; Mee, Robert P.; Morford, Gerald; Agrwal, Neera; Voss, Patricia G.; Moutsatsos, Ioannis K.; Wang, John L.

In: Gene, Vol. 60, No. 2-3, 1987, p. 197-204.

Research output: Contribution to journalArticle

Jia, Shizhe ; Mee, Robert P. ; Morford, Gerald ; Agrwal, Neera ; Voss, Patricia G. ; Moutsatsos, Ioannis K. ; Wang, John L. / Carbohydrate-binding protein 35 : molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli. In: Gene. 1987 ; Vol. 60, No. 2-3. pp. 197-204.
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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.",
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