CARBOHYDRATE-BINDING PROTEIN-35 .1. PROPERTIES OF THE RECOMBINANT POLYPEPTIDE AND THE INDIVIDUALITY OF THE DOMAINS

The cDNA clone for carbohydrate-binding protein 35 (CBP35) was enginee red into the bacterial expression vector pIN III ompA2, which directs the secretion of the expressed protein into the periplasmic space. Rec ombinant CBP35 was purified from this system, at a level of approximat ely 50 mg/liter of bacterial culture. Digestion of recombinant CBP35 w ith collagenase D, followed by purification using saccharide-specific affinity chromatography yielded a M(r) approximately 16,000 polypeptid e, corresponding to the COOH-terminal domain (residues 118-264) of the CBP35 polypeptide. This indicates that the COOH-terminal half of CBP3 5 contains the carbohydrate recognition domain, consistent with its se quence homology to other S-type lectins. The NH2-terminal domain (resi dues 1-137) was derived by site-directed mutagenesis of the cDNA, in w hich stop codons are inserted in place of Gly138 and Gly139, and expre ssion of the mutant cDNA in the same pIN III ompA2 system. The purifie d NH2-terminal domain failed to bind to saccharide-specific affinity r esins. Differential scanning calorimetry of rCBP35 and its individual domains yielded transition temperatures of approximately 39 and approx imately 56-degrees-C for the NH2- and COOH-terminal domains, respectiv ely. Lactose binding by the COOH-terminal domain shifted the transitio n temperature to 65-degrees-C, whereas sucrose failed to yield the sam e effect. These results suggest that the individual domains of the CBP 35 polypeptide are folded independently.