Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron.
We cloned the gene for one of the two chondroitin lyases of Bacteroides thetaiotaomicron into the cosmid vector pHC79 and subcloned it into pBR328. No proteins the size of B. thetaiotaomicron chondroitin lyase I or II (104 to 108 kilodaltons) were detectable in maxicell or in vitro transcription-translation preparations. However, partial purification of the chondroitin lyase activity from the Escherichia coli subclone showed that its properties were similar to those of the B. thetaiotaomicron chondroitin lyases. Antibodies to the chondroitin lyase that was produced in E. coli cross-reacted with the B. thetaiotaomicron chondroitin lyase II but not with chondroitin lyase I. The molecular weight of the enzyme produced in E. coli, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration, was slightly lower than those of the two chondroitin lyases from B. thetaiotaomicron; the enzyme had a higher affinity for bacterial membranes and for heparin-agarose, and cyanogen bromide digestion products of the chondroitin lyase produced in E. coli differed slightly from those of B. thetaiotaomicron chondroitin lyase II. gamma delta mutagenesis was used to locate the chondroitin lyase gene on the subcloned 7.8-kilobase EcoRI fragment. The size of the gene was approximately 3.3 kilobases, as expected for a protein with a molecular weight of 104,000.
D001439:Bacteroides / Q000201:enzymology / Q000235:genetics*; D002808:Chondroitin Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002810:Chondroitinases and Chondroitin Lyases / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D002808:Chondroitin Lyases; D002810:Chondroitinases and Chondroitin Lyases