Gene Cloning And Expression Of Creatinase Of Arthrobacter Nicotianae 02181

The determination of creatinine in biological fluids,which is an important factor in the evaluation of glomerular filtration function,is of great interest in clinical diagnosis.The creatinine level in blood serum and urine is clinically used as a parameter of renal damage. Creatinine is commonly determined enzymatically or by means of the Jaffe reaction both employing colorimetric indication.Although relatively cheap creatinine determinations in biofluids are possible with chemical method,which doesn't require expensive equipment and reagent,it has serious interference from several co-existing biological samples which called "false creatinine" and isn't well suited for clinical routine analysis at present. Therefore the development of rapid,sensitive and selective enzymatic method is promising and pressing need in clinical diagnosis.So far,the enzymatic method to determine creatinine in serum and urine is recommended by International Federation of Clinical Chemists(IFCC) and National Center for Clinical Laboratory(NCCL).There are three critical enzymes(creatininase,EC 3.5.2.10;creatinase,EC 3.5.3.3;sarcosine oxidase,EC 1.5.3.1) generally used in the enzymatic measurement system.Among these enzymes the creatinase is a very important one,which catalyzes the hydrolysis of creatine to form sarcosine and urea.The tool enzymes were mainly produced by several bacteria but having not been recombined in China until now.Costly imported kits for creatinine determination are used in most hospitals at present,so the development of recombined creatinase which we have the complete independent intellectual property right is critically important.Following is main contents and results of our research:1.Gene cloning of creatinase1.1 Cloning of partial sequence of creatinase gene7 sequences that from 7 different types of bacteria of all creatinase genes in GenBank were selected and submitted to the Block Maker service and 9 non-diastematic blocks were found.Degenerate primers had high scores and low degeneracy designed by the CODEHOP service according to these blocks were used in Degenerate PCR in which we got a 414-bp segment that was proved to be the partial of creatinase gene by BLASTx in NCBI which revealed high homology with those creatinase gene from different bacteria.1.2 Cloning of whole sequence of creatinase geneUpstream and downstream gene specific primers(GSP1 and GSP2) of the above sequence designed according to the GenomeWalker^TM Universal Kit were used in the method of genome walking to obtain the adjacent sequence of the known partial creatinase gene.The whole sequence of creatinase gene obtained by using the software BLASTx,Vector NTI suit 8 and Primer premier 5.0 consisted of 1254 bp(containing the stop codon TAA),and encoded 417 amino acid which had the theoretical molecular mass of 46377 Da. The result of BLASTx in NCBI showed that the identity and positive of amino acid was 79%(332/417) and 87%(365/417) respectively between the creatinase gene we got and that from Arthrobacter sp.FB24 among all those bacteria which produced creatinase.Primers with restriction enzyme sites(Eco RⅠand EK in the sense primer and SalⅠin the antisense primer) were designed based on the sequence and the creatinase gene was cloned by means of PCR.2.Expression of creatinase in E.coli BL21(DE3) and its purificationThe creatinase gene were digested with Eco RⅠand SalⅠand ligated to the vector pET42a which had been digested with the same enzymes.Then the recombined vector was transformed into E.coli DH5α.The positive clones confirmed by having been digested with the enzymes above and sequence mapping were transformed into E.coli BL21(DE3) to express the creatinase.The molecular mass of the protein produced by E.coli BL21(DE3) was estimated to be 67 kDa which matched the theory molecular mass well which contained the GST Tag(about 30 kDa) by comparing with standard proteins in the gel of SDS-PAGE. Then the GST Tag was cut out with EK during the primary GST purification and the "wild-type" creatinase(contained no other "foreign" amino acid) was obtained after the secondary GST purification.The molecular mass of the creatinase monomer was 46.4 kDa confirmed by SDS-PAGE.It was found that cell disruption by sonication resulted in a creatinase yield of 156.8 U/g cell,and a specific activity of 3.882 U/mg proteins,which was 14.7 and 24.7 folds than that produced by arthrobacter nicotianae 02181 respectively.The maximum activity of creatinase was exhibited at pH6.0 under the assay conditions at 37℃. The purified enzyme exhibited maximum activity at the temperature of 37℃,but the activity declined sharply while the temperature increased and it was nearly undetectable when incubated at 50℃for 30 min.About 40%of the original activity remained after incubated in phosphate buffer at 40℃for 30 min and it seemed to be stable at this level which indicated the enzyme might be used for clinical diagnosis.