Expression Of β-1,3-Glucanase Gene In Escherichia Coli

In this study the exo-β-1,3-glucanase gene(GenBank Accession No.X59259;EC No. 3.2.1.58) from Saccharomyces cerevisiae strain HS1185 was ligated with TA cloning vector pMDT-18 and transformed into Escherichia coli strain JM109.The constructed plasmid was named pMDT-18-GLU.Plasmid pMDT-18-GLU was digested with.Xho I and Nco I restriction enzymes and the obtained fragment ofβ-1,3-ghicanase gene was ligated with plasmid pET22b(+) at Xho I and Nco I restriction sites.The constructed plasmid was named pET22b-GLU.Plasmid pET22b-GLU was transformed into Escherichia coli strain BL21(DE3) and the positive colonies were identified by performing colony PCR.The extracted plasmid pET22b-GLU that contained the correct insert of theβ-1,3-glucanase gene and plasmid pET22b(+) was confirmed using PCR amplification and restriction enzyme analysis.The performed PCR gave amplified fragments of about 1337 bp-the size which was in accordance with the original size ofβ-1,3-glucanase gene from Sacchawmyces cerevisiae.For the restriction enzyme analysis,the recombinant plasmid pET22b-GLU was double digested with enzymes Xho I and Nco I and gave two bands of sizes 5431 bp and 1337 bp for plasmid pET22b(+) andβ-1,3-glucanase gene,respectively.Further confirmation of the correct insert was done by carrying out DNA sequencing of the cloned gene.The nucleotide sequence results showed high identity of 98%to the original beta-l,3-glucanase gene from Saccharomyces cerevisiae.The recombinant Escherichia coli strain BL21(DE3) carrying plasmid pET22b-GLU was cultivated and induced with IPTG for protein expression.The results showed that there was no activity using the DNS assay for reducing sugar.However,based on the results obtained,β-l,3-glucanase gene from Saccharomyces cerevisiae strain HS185 was successfully cloned into plasmid pET22b(+) and transformed in Escherichia coli strain BL21(DE3).