Parallel gene cloning and protein production in multiple expression systems.

To quickly find an optimal expression system for recombinant protein production, a set of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously. These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5'-EcoRI/3'-XhoI identical in these vectors for ligating with the sticky-end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort.

Structural basis for catalytic and inhibitory mechanisms of human prostaglandin reductase PTGR2.

PTGR2 catalyzes an NADPH-dependent reduction of the conjugated alpha,beta-unsaturated double bond of 15-keto-PGE(2), a key step in terminal inactivation of prostaglandins and suppression of PPARgamma-mediated adipocyte differentiation. Selective inhibition of PTGR2 may contribute to the improvement of insulin sensitivity with fewer side effects. PTGR2 belongs to the medium-chain dehydrogenase/reductase superfamily.

An improved SUMO fusion protein system for effective production of native proteins.

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins.

Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex.

Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm.

Self-cleavage of fusion protein in vivo using TEV protease to yield native protein.

Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins.

Characterization of the L399P and R447G mutants of hsc70: the decrease in refolding activity is correlated with an increase in the rate of substrate dissociation.

It is known that 70-kDa heat-shock cognate protein (hsc70) is capable of forming complexes with unfolded polypeptide substrates and works with DnaJ homologues to refold denatured proteins. Herein, we characterized two hsc70 mutants, hsc70(L399P) and hsc70(R447G). They retained the capability of restoring the activity of denatured luciferase, but their activity was decreased to 40% and 20%, respectively, of that of hsc70.