Unpredictive Expression of recombinant proteins under the control of different promoters in different expression systems

Abstract

Synthetic human proinsulin gene with E.coli preferred codon and extra methionine was expressed as 30% of total cellular protein. Fusing seven aminoacids of well expressed protein to the N’-terminus did not alter the expression of recombinant proinsulin. Doubling the copies of proinsulin gene will not doubles the expression. The protein was expressed as inclusion bodies. Changing the distances between ribosome binding site and start codon effect the expression in an unexplained way. Ten nucleotide distance gave expression of 2-4% and with 12 nucleotides, expression decreasaes up to 1-2%.. Incorporating 10 different nucleotides at the start of proinsulin gene decreases the mRNA secondary structure but did not alter the expression. Metabolic instability, rapid degradation of mRNA or accumulation of protein may down regulate expression of mRNA. High expression of azurin gene in pET22b vector was obtained when fused with 19 aminoacid signal peptide in BL21-Codon Plus (DE3) cells. PE38KDEL can be expressed in huge quantities in Escherichia coli by using the recombinant vector PE38KDEL/pET under control of T7 promoter and E. coli host strain BL21 (DE3) CodonPlus. The protein expressed as soluble and insoluble inclusion bodies the expression in soluble form was more pronounced. When human interferone alpha2|B gene and E.coli amino peptidase genes were expressed bicistronically in expression plasmid pET-21a (+). There was negligible expression of human interferone alpha 2B. PelB fused DNA polymerase I was expressed as soluble fraction with maximum expression with 0.5mM IPTG and 20mM lactose. Recombinant protein expression in expression host can be improved by mRNA stability, codon bias, and controlling inclusion bodies formation and changing genetic tools in different combinations. There are other controlling factors of protein expression i.e., metabolic instability, rapid degradation of mRNA or accumulation of protein may downregulate expression of mRNA.