CLONING, EXPRESSION, AND SPECTROSCOPIC STUDIES OF THE JUN LEUCINE-ZIPPER DOMAIN

Association of the human c-Jun and c-Fos proteins depends upon interac tions involving their leucine zipper domains. We are interested in elu cidating the tertiary structure of the Jun and Fos leucine zipper doma ins with a view to understanding the precise intermolecular interactio ns which govern the affinity and specificity of interaction in these p roteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both un labelled and isotopically labelled c-Jun leucine zipper domain. A synt hetic junLZ gene was created by annealing, ligation, and polymerase-ch ain-reaction amplification of overlapping synthetic oligonucleotides w hich comprised 132 bp of coding sequence encompassing residues Arg276- Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini. The junLZ gene was cloned into the pGEX-2T vect or from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 resi dues, 5.1 kDa) was overexpressed (approximate to 15% total cell protei n) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione S-transferase. Two markedly different expression strategie s have been devised which allow purification of rJunLZ from the solubl e or inclusion-body fraction of induced cells. We have used these stra tegies to produce unlabelled and uniformly N-15-labelled rJunLZ for NM R studies which, in combination with circular dichroic measurements, r eveal that rJunLZ most likely forms a symmetric coiled-coil of paralle l alpha-helices. We also present N-15-NMR chemical shift assignments f or the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodime ric Jun leucine zipper using heteronuclear three-dimensional NMR spect roscopy.