CLONING AND EXPRESSION OF ClpD, A CHLOROPLASTIC CHAPERONE FROM Arabidopsis thaliana

ROSANO, G. L.; CECCARELLI, E. A.

Rosario, Argentina

Congreso; XLII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular 2 al 15 de Noviembre de 2006 - Centro Cultural Parque de España; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Chaperones of the Hsp100 family (ClpC1/C2/D) have beenimplicated in protein folding and in the translocation ofpolypeptides in chloroplasts, events of great importance fornormal plant growth. ClpD is the only one expressed under lightand cold stress; therefore, could be the main chaperoneparticipating in the protein quality control machinery ofchloroplasts and/or the translocation of chloroplastic proteinsunder these adverse conditions. The goal of this work is to gainfurther insight into the role of ClpD in the aforementionedprocesses. The cDNA of the gene clpd was obtained from aArabidopsis thaliana cDNA bank. A 2650 bp product wasamplified by PCR and cloned in plasmid pET28a, in order to tagthe protein with histidines. The construct was transformed intoEscherichia coli cells; then, the expression of ClpD was assayed.After cellular lysis, the soluble and insoluble fractions wereanalyzed by SDS-PAGE and Western blot using anti-Hisantibodies. Under usual expression conditions, the protein waslocated in inclusion bodies. Thus, the expression was optimizedby varying IPTG concentration, induction temperature andinduction length. Under all conditions, a band of 98 kDa wasobserved. Under very mild expression conditions, a 60% ofsoluble protein was recovered and, by the use of affinitycolumns, purified. Its folding state and activity were alsoanalyzed.