. Influence of RBS-ATG distance and mRNA structure in the optimal expression using pET-22B(+)/ BL21 system

MARIA JULIA OUSSET; BELAICH MARIANO; BILEN MARCOS; LOZANO MARIO ENRIQUE; GHIRINGHELLI DANIEL

Carlos Paz

Congreso; XXXVII Annual Meeting of the Argentine Society for the Biochemistry and Molecular Biology Research; 2001

Heterologous systems for protein expression are one of the most important tools in the molecular biology laboratories and biotechnology industries. Milligrams of some proteins are necessary to produce monoclonal and policlonal antibodies, to perform functional and structural studies and a lot of another possible applications. Eukaryotic and prokaryotic systems have been developed to produce high levels of recombinant proteins. The pET-22b(+)/E. coli BL21-DE3 prokaryotic system is one of the most utilized, because it could produce very high levels of proteins at low cost. However, successful cloning and expression using this system depend of the construct architecture. In particular, the optimal RBS-ATG distance must not exceed 12 nucleotides, and the mRNA 5’ end secondary structure must allow the ribosome assembly and finding the ATG codon. In this work, we show the cloning and protein expression of one baculoviral gene, the Anticarsia gemmatalis MNPV polyhedrin gene, using the pET-22b(+)/E. coli BL21-DE3 prokaryotic system. We analyzed the influence of RBS-ATG distance and mRNA 5’ end secondary structure using different constructs, with the goal to state the optimal conditions to obtain the best expression levels of recombinant protein.