A plant transit peptide directs proteins to the Hsp100 chaperone system

BRUCH EDUARDO MARCOS; ROSANO, GERMÁN L; CECCARELLI, EDUARDO A

Tucumán, Argentina

Congreso; XLV Reunión Anual SAIB; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Transit peptides (TP) are N-terminal extensions that route nuclear encoded proteins into plastids. Although Hsp100 chaperones have been implicated in precursor import into plastids, evidences for their interaction with TPs are lacking. Two plasmids were constructed for the expression of green fluorescent protein (GFP) and an amino terminal plant TP fusion to GFP, both with a six-histidine amino-terminal tag (His-GFP and His-TP-GFP). The constructs were transformed into  Escherichia coli cells. The expression and purification conditions for both of them were assayed. After cellular lysis, the soluble fractions were purified using a Ni-NTA-Agarose resin. Products were analyzed by SDS-PAGE and Western blot using anti-His and anti-GFP antibodies. Under usual expression conditions (25 °C for 4 hs, 0.5 mM IPTG), His-GFP was purified satisfactorily but His-TP-GFP was not. Thus, the expression was optimized. Optimal conditions for expression were achieved at 0.5 mM IPTG, 18 °C for 16 hs and then, purifying the protein at 0 °C. Pull Down analyses were carried out using these two proteins as baits. Quantification of the inmunoblots showed that the His-TP-GFP retained about sixty times more ClpA (an  E. coli Hsp100 chaperone) than His-GFP. These observations indicate the existence of an interaction between a plant TP and an Hsp100 protein from E. Coli.