A method for the screening of NADP-malic enzyme activity in bacteria colonies

ALVAREZ, C.E.; DETARSIO, E.; SAIGO, M; ANDREO, C.S.; DRINCOVICH, M.F.

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, SAIB

Random replacement of residues in protein sequences and selective studies of new variants, which is commonly known as in vitro evolution, represent a useful strategy for the identification of residues that confer special kinetic and/or structural properties. The key point in this approach is the screening of a large number of clones in order to identify those that have a special feature such as higher catalytic efficiency, resistance to high temperatures or denaturing agents, among others. The aim of this work was to develop an assay to detect catalytic activity of NADP dependent Malic Enzyme (NADP-ME) in a large number of E. coli colonies within a reasonable period of time. The strategy consists of fixing the proteins in a solid support to facilitate the detection of activity in vivo. To reach this purpose, the colonies transformed were absorbed by a nitrocellulose filter and transferred to LB plates. After overnight incubation, when the colonies had reached the same size, the cell walls were lysed and the proteins fixed to the membrane to evaluate enzymatic activities. This assay allows us to recognize recombinant proteins with different levels of activity minimizing the interference of background endogenous activity. To resume, it is remarkable to notice that this technique is an essential tool for the screening of new versions of malic enzymes