EXPRESSION AND CHARACTERIZATION OF UDPGLC DEHYDROGENASE (KFID), WHICHIS ENCODED IN THE TYPE-SPECIFIC REGION-2 OF THE ESCHERICHIA-COLI K5 CAPSULE GENES

Region 2 of the Escherichia coli K5 capsule gene cluster contains four genes (kfiA through -D) which encode proteins involved in the synthes is of the K5 polysaccharide. A DNA fragment containing kfiD was amplif ied by PCR and cloned into the gene fusion vector pGEX-2T to. generate a GST-KfiD fusion protein. The fusion protein was isolated from the c ytoplasms of IPTG (isopropyl-beta-D-thiogalactopyranoside) induced rec ombinant bacteria by affinity chromatography and cleaved with thrombin , The N-terminal amino acid sequence of the cleavage product KfiD' cor responded to the predicted amino acid sequence of KfiD with an N-termi nal glycyl-seryl extension from the cleavage site of the fusion protei n. Anti-KfiD antibodies obtained with KfiD' were used to isolate the i ntact KfiD protein from the cytoplasms off. coli organisms overexpress ing the kJiD gene. The fusion protein, its cleavage product (KfiD'), a nd overexpressed KfiD converted UDPGlc to UDPGlcA. The KfiD protein co uld thus be characterized as a UDPglucose dehydrogenase.