Poster presentation: Electrophoretic assessment of recombinant λ- exonuclease production in different E. coli strains

Abstract

Viral exonucleases play role in many processes essential for genome ma intenance,including DNA repair and recombination. Lambda exonuclease (λ -exo), isolated fromlambda bacteriophage, hydrolases double-stranded DNA (dsDNA) in the highly processivemanner in 5’→3’ direction, yielding mononucleotides and single -stranded DNA (ssDNA).This unique enzymatic properties offer several promising biotechnological applications,such as highly sensitive quantification of DNA modifications and single -moleculesequencing. Hence, optimization of the expression conditions is a prerequisite to achievehigh-level production of λ-exo. Here we have tested λ -exo expression in five different E.coli strains under various temperature regimes in order to establish the optimal conditionsfor efficient production of recombinant λ -exo. The N-terminally His -tagged λ-exo wassuccessfully expressed in E.coli BL21(AI), SHuffle T7, C41(DE3) and C43(DE3) strains inLB broth. Collected aliquots were analysed by SDS-PAGE, followed by CBB staining.Relative yield of target protein bands was determined by densitometry in total cell lysate, aswell as in soluble and insoluble cytoplasmatic fractions. We identified E.coli BL21(AI),SHuffle T7 and C41(DE3) as good producers of recombinant λ -exo, and upon scaling up, λ-exo was purified from crude cell lysates by metal affinity chromatography in satisfactoryyield. Our data suggest that densitometric analysis could serve as a powerful low-costscreening platform for improving recombinant protein expression strategies.