ABSTRACT
As internal protein elements , inteins are self-excised from their host protein and catalyze ligation of exteins with a peptide bond .They are widely found in all the three domains of the biological kingdom , and in viral proteins .Intein-mediated protein splicing is a posttranslational autocatalytic process that does not require auxiliary enzymes or cofactors . Protein splicing involves four intramolecular reactions and a small number of key catalytic residues in the intein and exteins . The development of expressed protein ligation ( EPL ) and protein trans-splicing ( PTS ) is assisted by inteins and protein splicing.This review introduces the biosynthesis of backbone-cyclized peptides libraries , and describes the applications of cyclic polypeptides and their prospective applications in the future .
ABSTRACT
Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO(small ubiq-uitin-related modifier) using the widely used solubility promoting protein SUMO and based on the translational coupling phenomenon in order to enable the non-fusion soluble expression of the broad-spectrum antiviral protein RA in Escherichia coli by pTIG-mSUMO.Methods The smt3 gene coding for SUMO protein was cloned from yeast genome DNA by PCR. After directed-site silent mutation to eliminate the EcoRⅠsite, the mutant mSUMO was inserted into pET-22b to obtain the translational coupling expression vector pTIG-mSUMO.The RA was subject to PCR amplification and cloned into the pTIG-mSUMO to obtain the expression plasmid pTIG-mSUMO/RA which was supposed to direct the soluble expression of RA by the translational coupling with mSUMO.Results A translational coupling expression vector pTIG-mSUMO which could di-rect/drive the SUMO and heterogonous protein non-fusion expression simultaneously was designed and constructed.The Western blotting result indicated that pTIG-mSUMO could direct the high-level expression of RA, around 40%of which was soluble.Conclusion A translational coupling expression vector pTIG-mSUMO is obtained.After coupling with SUMO, RA is highly expressed in E.coli and both the expression level and solubility are greatly improved.pTIG-mSUMO might contrib-ute to soluble expression of other proteins.
ABSTRACT
We reconstructed the erythromycin macrocyclic lactone (6-deoxyerythronolide B, 6dEB) synthesis pathway in Escherichia coli. We first cloned all the genes needed to synthesize the 6dEB into multi-gene co-expressed vectors. Then using the recognition sequences of isoschizomers Xba I/Spe I of vectors, we assembled the related genes into a series multiple-genes recombinant plasmids pBJ144, pBJ130. The recombinant plasmids pBJ144, pBJ130 were cotransformed into BAP1 to get the recombinant BAP1(pBJ144/pBJ130). SDS-PAGE analysis showed that individual genes were expressed correctly. After inducing at low temperature, adding propionate as substrate, we validated the crude product by mass spectrometry and the 6dEB yield was about 10 mg/L. These results indicated that the synthetic pathway of 6dEB was successfully assembled and reconstructed in Escherichia coli, which will greatly facilitate the reconstruction of whole erythromycin synthesis pathway and finally help to establish a stable research platform for developing of new derivatives of erythromycin and combinatorial biosynthesis of polyketide-type antibiotics.
Subject(s)
Anti-Bacterial Agents , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Erythromycin , Escherichia coli , Genetics , Metabolism , Genetic Vectors , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To observe the effects of Qufengtongluo Recipe (QFTLR) on the expressions of cell cycle regulatory proteins in rat mesangial cells (MCs) in vitro and investigate the mechanism by which QFTLR inhibits MC proliferation.</p><p><b>METHODS</b>Using the methods of serum pharmacology, we studied the expressions of cell cycle regulatory proteins in rat MCs treated with QFTLR by laser scanning confocal microscope and immunohistochemistry.</p><p><b>RESULTS</b>Compared with the normal control cells, the cells challenged with lipopolysaccharide (LPS) showed significantly enhanced expressions of cyclin D1, CDK2, and P21 (P<0.01) and obviously lowered protein expression of P27 (P<0.01). Treatment of the LPS-challenged cells with QFTLR and benazepril both resulted in significantly attenuated expressions of cyclin D1, CDK2, and P21 and obvious increase of P27 expression (P<0.05 or P<0.01), but QFTLR produced stronger effects than benazepril in regulating of cyclinD1, P21 and P27 protein expressions (P<0.05 or P<0.01).</p><p><b>CONCLUSION</b>QFTLR inhibits rat MC proliferation in vitro possibly by down-regulating the cellular expressions of cyclin D1, CDK2, and P21 and up-regulating the expression of P27 protein.</p>