Submitted to: Gene Expression
Publication Type: Abstract Only
Publication Acceptance Date: 3/6/1996
Publication Date: N/A
Citation: Interpretive Summary:
Technical Abstract: The Stress70 molecular chaperone "machine" is composed of multiple protein components. In E. coli the machine contains products of the dnaK, dnaJ and grpE genes. DnaK recognizes and binds to unfolded proteins, and is characterized by a low-level, protein or peptide-stimulated ATPase activity. DnaJ stimulates hydrolysis of ATP by DnaK and, in combination with the nucleotide exchange factor GrpE, increases the ATPase activity many-fold. We have sequenced a cDNA for a higher plant (Arabidopsis thaliana) homologue of dnaJ, atJ2. The deduced amino acid sequence of AtJ2 is 48% homologous (identity plus similarity) with E. coli DnaJ, and 58% homologous with the Saccharomyces homologue, YdJ1. Recombinant AtJ2, produced as a chimera with the Maltose Binding Protein in E. coli, is capable of stimulating the ATPase activity of both bacterial DnaK and its plant homologues. In order to study interactions among AtJ2, target polypeptides, and other chaperone proteins in vitro, we wished to produce large quantities of recombinant AtJ2. A vector was constructed based upon the Pichia expression plasmid, pHIL-D2. The modified vector has a synthetic double-stranded oligonucleotide inserted into the EcoRI site of pHIL-D2, making several additional restriction sites are unavailable. Using this vector, the AtJ2 protein was expressed with an N-terminal His6 followed by a thrombin cleavage site and the mature protein N-terminus. From the protease minus host strain SMD1168, we were able to obtain substantial levels of homogenous recombinant AtJ2.