ABSTRACT
Objective To clone express and purify Schistosoma japonicum fructose?1 6?bisphosphate aldolase SjFBPA in E. coli and observe its expression in different developmental stages of S. japonicum. Methods FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid and inducibly expressed with IPTG in E. coli BL21. SDS?PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA rSjFBPA . Then rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS?PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore SjFBPA mRNA was ana?lyzed in different developmental stages of S. japonicum by RT?PCR. Results SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro?tein could specifically reactive to the anti?His?tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT?PCR showed that SjFBPA mRNA was expressed in cercaria schistosomulum adult worm and egg of S. japonicum. Conclusion SjFBPA is successfully recombined and expressed in a prokaryotic system and SjFBPA mRNA is expressed in cercaria schistosomulum adult worm and egg of S. japonicum.
ABSTRACT
The complete genomes of more cyanobacterial strains have been completed for sequence, and genetic engineering of cyanobacteria has evolved in the post-genome era. Since Kaneko and colleagues had completed the sequence for the complete genome of Anabaena sp. PCC 7120 in 2001, functions of some genes in this genome, including fructose-1,6-bisphosphate aldolase (FBA) gene, have been predicated using the method of bioinformatics. However, little information is available regarding whether this gene can encode FBA and its product characteristics of related enzyme. Here, to explore this information, the predicted II-FBA gene-encoding region in Cyanobase database was cloned by PCR method and then ligated into pET-32a to generate the expression vector, pET-FBA-II. The results of SDS-PAGE indicated that the expression level of the expected target polypeptide was approximate 23.4 percent as compared to total protein and the molecular weight is about 40 kDa as compared to the protein molecular marker. The results of enzyme activity analysis showed that the activity of II-FBA was ~11.8 U per mg protein and owned a standard activity of II-FBA. To sum up, the results not only prove the functional prediction of this II-FBA gene from the Cyanobase database, but also provide the important conditions for further studying its physiological and biochemical characteristics and functions of the gene expression product.
ABSTRACT
Fructose 1,6-bisphosphate aldolase (FPA) was recently known as new member of heparin binding proteins and a new method for FPA purification has been proposed (Thanh Van Ta et all, J. Biochem. 125, 554-559,199) by measuring FPA - heparin binding inhibition caused by various glycosaminoglycans (GAGs), affinity of the two isoforms, aldolase A4 and C4, to the GAGs underphysiological ionic conditions was estimated. Among glycosaminoglycans employd, heparin was confirmed to be the unique one that could bind specifically these enzymes. In the lower ionic strength, the affinity order of both FPA isoforms (A4 and C4) to these GAGs appeared as heparin> chondroitin polysulfate> heparin sulfate > dermatan > chondrointin sulfate A > chondroin sulfate C. Employing the same techniques, the affinity of regioselectively desulfated heparins to FPA was estimated. Our results indicated that, among the sulfate groups is heparin, loss of N-sulfate group reduced most significantly the affinity to FPA A4 and C4. This sugests that FPA recognizes a specific heparin structure including the sulfo-amino group at C2 of the glucosamine residue as the vital factor in this interaction.