Cloning and Production of recombinant maize cyclin D6 protein

LB PENA; E GARCIA RAMIREZ; AAE MENDEZ; ME VINACOUR; JM VAZQUEZ RAMOS; SM GALLEGO

Congreso; L Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2014

Cyclins are primary regulators of the activity of cyclin-dependent kinases and play crucial roles in cell cycle progression in eukaryotes. In plants, D-type cyclins (G1 cyclins) regulate cell cycle reentry during meristem activation to promote successful germination and early seedling growth. Moreover, the larger number of cyclin D (CYCD) genes in plants compared with animals is indicative of the importance of these proteins being involved in the linkage between environmental conditions and the plant cell cycle. As part of a project that study posttranslational modifications (MPT) of proteins related to cell cycle in plants, herein we present the cloning and expression of one of the Zea mays L (maize) cyclin D -Zeama;CycD6;1- characterized by lacking of the pRBR-binding sequence-. The ADN fragment corresponding to CycD6 was isolated using specific primers and cloned in a pGEX 4T-2. Initially BL21-DE3 E. coli strain was chosen as expression system. Since the level of recombinant protein produced by this system was low and mainly located in inclusion bodies, the plasmid was transferred to Rosetta gami pLysS strain. This system resulted in a significant improvement regarding the levels of recombinant protein production. Through analysis using informatics tools an approximation of the protein sites susceptible to suffer MPT will be presented.