Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains.
The cDNA clone for carbohydrate-binding protein 35 (CBP35) was engineered into the bacterial expression vector pIN III ompA2, which directs the secretion of the expressed protein into the periplasmic space. Recombinant CBP35 was purified from this system, at a level of approximately 50 mg/liter of bacterial culture. Digestion of recombinant CBP35 with collagenase D, followed by purification using saccharide-specific affinity chromatography yielded a M(r) approximately 16,000 polypeptide, corresponding to the COOH-terminal domain (residues 118-264) of the CBP35 polypeptide. This indicates that the COOH-terminal half of CBP35 contains the carbohydrate recognition domain, consistent with its sequence homology to other S-type lectins. The NH2-terminal domain (residues 1-137) was derived by site-directed mutagenesis of the cDNA, in which stop codons are inserted in place of Gly138 and Gly139, and expression of the mutant cDNA in the same pIN III ompA2 system. The purified NH2-terminal domain failed to bind to saccharide-specific affinity resins. Differential scanning calorimetry of rCBP35 and its individual domains yielded transition temperatures of approximately 39 and approximately 56 degrees C for the NH2- and COOH-terminal domains, respectively. Lactose binding by the COOH-terminal domain shifted the transition temperature to 65 degrees C, whereas sucrose failed to yield the same effect. These results suggest that the individual domains of the CBP35 polypeptide are folded independently.
Agrwal
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Department of Biochemistry, Michigan State University, East Lansing 48824.
Sun
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Wang
S Y
SY
Wang
J L
JL
D000595:Amino Acid Sequence; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002853:Chromatography, Liquid; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D037502:Galectin 3; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000943:Antigens, Differentiation; D037502:Galectin 3; D011994:Recombinant Proteins; D004247:DNA