A plant transit peptide directs the hsp100 chaperone system

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

San Miguel de Tucumán, Tucumán

Congreso; XLV Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB); 2009

Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB)

Transit peptides (TP) are N-terminal extensions that route nuclearencoded proteins into plastids. Although Hsp100 chaperones havebeen implicated in precursor import into plastids, evidences for theirinteraction with TPs are lacking. Two plasmids were constructed forthe expression of green fluorescent protein (GFP) and an aminoterminal plant TP fusion to GFP, both with a six-histidine aminoterminaltag (His-GFP and His-TP-GFP). The constructs weretransformed into Escherichia coli cells. The expression andpurification conditions for both of them were assayed. After cellularlysis, the soluble fractions were purified using a Ni-NTA-Agaroseresin. Products were analyzed by SDS-PAGE and Western blot usinganti-His and anti-GFP antibodies. Under usual expressionconditions (25 °C for 4 hs, 0.5 mM IPTG), His-GFP was purifiedsatisfactorily but His-TP-GFP was not. Thus, the expression wasoptimized. Optimal conditions for expression were achieved at 0.5mM IPTG, 18 °C for 16 hs and then, purifying the protein at 0 °C.Pull Down analyses were carried out using these two proteins asbaits. Quantification of the inmunoblots showed that the His-TPGFPretained about sixty times more ClpA (an E. coli Hsp100chaperone) than His-GFP. These observations indicate the existenceof an interaction between a plant TP and an Hsp100 protein from E.Coli.