Genomic sequencing and recombinant expression of proinsulin isolated from cow and buffalo in Pakistan

This study was conducted to determine the heterologous expression of eukaryotic gene in Escherichia coli variable. The expression of Pakistani buffalo (Bubalus bubalis) and cow (Bos taurus) proinsulin genes in BL21 codon plus cells is 30% of total cellular protein. Total RNA was isolated from pancreatic tissues of local (Pakistani) breeds of buffalo (B. bubalis) and cow (B. taurus), converted to cDNA and cloned in a T/A cloning vector.  There are five silent mutations: one in B-chain, two each in A-chain and C-peptide encoding regions of local Pakistani buffalo proinsulin DNA sequence as compared to the internationally reported cow proinsulin sequence. The DNA sequence of local (Pakistani) cow proinsulin shows there is one nucleotide encodes C-peptide that has mismatch with the reported cow proinsulin sequence but mismatched nucleotide is same between Pakistani cow and Pakistani buffalo proinsulin sequence. This indicates the genetic similarity of Pakistani cow with Pakistani buffalo.  Both genes were expressed in BL21 Codon plus (DE3)-RIL cells with 0.2 mM IPTG at 37°C for 6 to 8 h. Proinsulin was expressed as 30% of total cellular proteins as insoluble inclusion bodies.   Key words: Bubalus bubalis, Bos taurus, cDNA, T/A cloning vector, BL21 Codon plus (DE3)-RIL cells.