Open AccessCloning, expression, and spectroscopic studies of the Jun leucine zipper domain
Author(s) -
RILEY Lisa G.,
JUNIUS F. Keith,
SWANTON Michael K.,
VESPER Natalie A.,
WILLIAMS Neal K.,
KING Glenn F.,
WEISS Anthony S.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18569.x
Subject(s) - leucine zipper , bzip domain , zipper , coiled coil , basic helix loop helix leucine zipper transcription factors , atf3 , biochemistry , expression vector , biology , fusion protein , gene , oligonucleotide , cloning (programming) , chemistry , recombinant dna , microbiology and biotechnology , peptide sequence , transcription factor , gene expression , dna binding protein , promoter , algorithm , computer science , programming language
Association of the human c‐Jun and c‐Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, 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 unlabelled and isotopically labelled c‐Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase‐chain‐reaction amplification of overlapping synthetic oligonucleotides which 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 pGEX2T vector from which recombinant c‐Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (∼15% total cell protein) 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 strategies have been devised which allow purification of rJunLZ from the soluble or inclusion‐body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15 N‐labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled‐coil of parallel α‐helices. We also present 15 N‐NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high‐resolution structure of the homodimeric Jun leucine zipper using heteronuclear three‐dimensional NMR spectroscopy.