Cloning and expression of ClpD, a Chloroplastic chaperone from Arabidopsis thaliana

ROSANO, GERMÁN L.; CECCARELLI, EDUARDO A

Rosario

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Chaperones of the Hsp100 family (ClpC1/C2/D) have been implicated in the assistance of protein folding and the translocation of polypeptides in chloroplasts, events of great importance for normal plant growth. ClpD is the only one expressed under light and cold stress; therefore, could be the main chaperone participating in the protein quality control machinery of chloroplasts and/or the translocation of chloroplastic proteins under these adverse conditions. The goal of this work is to gain further insight into the role of ClpD in the aforementioned processes. The cDNA of the gene clpd was obtained from a Arabidopsis thaliana cDNA bank. A 2650 bp product was amplified by PCR and cloned in plasmid pET28a, in order to tag the protein with histidines. The construct was transformed into Escherichia coli cells; then, the expression of ClpD was assayed. After cellular lysis, the soluble and insoluble fractions were analyzed by SDS-PAGE and Western blot using anti-His antibodies. Under usual expression conditions, the protein was located in inclusion bodies. Thus, the expression was optimized by varying IPTG concentration, induction temperature and induction length. Under all conditions, a band of 98 kDa was observed. Under very mild expression conditions, a 60% of soluble protein was recovered and, by the use of affinity columns, purified. Its folding state and activity were also analyzed.