Solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive Escherichia coli protein, RpoS

<p>Abstract</p> <p>Background</p> <p>The most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. Although commercial fusion partners including maltose binding protein and glutathione-<it>S</it>-transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. Thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study.</p> <p>Results</p> <p>We analyzed the <it>Escherichia coli </it>proteome response to the exogenous stress of guanidine hydrochloride using 2-dimensional gel electrophoresis and found that RpoS (RNA polymerase sigma factor) was significantly stress responsive. While under the stress condition the total number of soluble proteins decreased by about 7 %, but a 6-fold increase in the level of RpoS was observed, indicating that RpoS is a stress-induced protein. As an N-terminus fusion expression partner, RpoS increased significantly the solubility of many aggregation-prone heterologous proteins in <it>E. coli </it>cytoplasm, indicating that RpoS is a very effective solubility enhancer for the synthesis of many recombinant proteins. RpoS was also well suited for the production of a biologically active fusion mutant of <it>Pseudomonas putida </it>cutinase.</p> <p>Conclusion</p> <p>RpoS is highly effective as a strong solubility enhancer for aggregation-prone heterologous proteins when it is used as a fusion expression partner in an <it>E. coli </it>expression system. The results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase.</p

Forward-central two-particle correlations in p-Pb collisions at root s(NN)=5.02 TeV

Two-particle angular correlations between trigger particles in the forward pseudorapidity range (2.5 2GeV/c. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B. V.Peer reviewe

Event-shape engineering for inclusive spectra and elliptic flow in Pb-Pb collisions at root(NN)-N-S=2.76 TeV

Peer reviewe

Elliptic flow of muons from heavy-flavour hadron decays at forward rapidity in Pb-Pb collisions at root s(NN)=2.76TeV

The elliptic flow, v(2), of muons from heavy-flavour hadron decays at forward rapidity (2.5 <y <4) is measured in Pb-Pb collisions at root s(NN)= 2.76TeVwith the ALICE detector at the LHC. The scalar product, two- and four-particle Q cumulants and Lee-Yang zeros methods are used. The dependence of the v(2) of muons from heavy-flavour hadron decays on the collision centrality, in the range 0-40%, and on transverse momentum, p(T), is studied in the interval 3 <p(T)<10 GeV/c. A positive v(2) is observed with the scalar product and two-particle Q cumulants in semi-central collisions (10-20% and 20-40% centrality classes) for the p(T) interval from 3 to about 5GeV/c with a significance larger than 3 sigma, based on the combination of statistical and systematic uncertainties. The v(2) magnitude tends to decrease towards more central collisions and with increasing pT. It becomes compatible with zero in the interval 6 <p(T)<10 GeV/c. The results are compared to models describing the interaction of heavy quarks and open heavy-flavour hadrons with the high-density medium formed in high-energy heavy-ion collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V.Peer reviewe

Measurement of charged jet production cross sections and nuclear modification in p-Pb collisions at root s(NN)=5.02 TeV

Charged jet production cross sections in p-Pb collisions at root s(NN) = 5.02 TeV measured with the ALICE detector at the LHC are presented. Using the anti-k(T) algorithm, jets have been reconstructed in the central rapidity region from charged particles with resolution parameters R = 0.2 and R = 0.4. The reconstructed jets have been corrected for detector effects and the underlying event background. To calculate the nuclear modification factor, R-pPb, of charged jets in p-Pb collisions, a pp reference was constructed by scaling previously measured charged jet spectra at root s = 7 TeV. In the transverse momentum range 20Peer reviewe

Masking thiol reactivity with thioamide, thiourea, and thiocarbamate-based MBPs.

Thioamides, thioureas, and thiocarbamates are introduced as stable, sulfur-based metal-binding pharmacophores (MBPs) for use in metalloenzyme fragment-based drug discovery (mFBDD). MBP reactivity, bioactivity, and structural studies show that these molecules can act as ligands for Zn(II)-dependent metalloenzymes including human carbonic anhydrase II (hCAII) and matrix metalloproteinase-2 (MMP-2)

Functional fusion mutant of Candida antarctica lipase B (CalB) expressed in Escherichia coli

Candida antarctica lipase B (CalB) was functionally expressed in the cytoplasm of Escherichia coli Origami (DE3) with the N-terminus fusion of E. coli endogenous proteins. The previously-identified stress responsive proteins through comparative proteome analyses such as malate dehydrogenase (Mdh), spermidine/putrescine-binding periplasmic protein (PotD), and FKBP-type peptidyl-prolyl cis-trans isomerase (PPlases) (SlyD) dramatically increased the solubility of CalB in E coli cytoplasm when used as N-terminus fusion partners. We demonstrated that Mdh, PotD, and SlyD were powerful solubility enhancers that presumably facilitated the protein folding of CalB. Moreover, among the various fusion mutants, Mdh-CalB showed the highest hydrolytic activity and was as biologically active as standard CalB. Similarly to the previous report, the electrophoretic properties of CalB indicate that CalB seems to form dimer-based oligomer structures. We evaluated the structural compatibility between the fusion partner protein and CalB, which seems to be of crucial importance upon the bioactive dimer formation of CalB and might affect the substrate accessibility to the enzyme active site, thereby determining the biological activities of the fusion mutants.clos