Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression

Unger, Tamar; Jacobovitch, Yossi; Dantes, Ada; Bernheim, Reut; Peleg, Yoav

doi:10.1016/j.jsb.2010.06.016

Cited by 224 publications

(176 citation statements)

References 27 publications

Supporting

Mentioning

176

Contrasting

Order By: Relevance

“…The plasmids used for protein expression and chromosome editing are listed in supplemental Table S2. The pBAD-SurA-His 6 , pBAD-OmpF-His 6 , and pBAD-LamB-His 6 plasmids were constructed by inserting the genes (amplified from the genomic DNA of Escherichia coli cells) encoding SurA, OmpF, or LamB into the pBAD/Myc-His C vector using the restriction enzyme free cloning (RF-cloning) method (32). Site-directed mutagenesis was performed using the Phusion site-directed mutagenesis kit (New England Biolabs).…”

Section: Methodsmentioning

confidence: 99%

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

Wang

Feng

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

␤-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent ␤-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent ␤-barrel OMPs largely via its N-domain but with ␤-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent ␤-barrel OMPs interact with SurA via their N-and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving ␤-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for ␤-barrel OMP biogenesis.

show abstract

Section: Methodsmentioning

confidence: 99%

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

Wang

Feng

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Recombinant laccases were cloned by using a two-step restrictionfree procedure (63). The plasmids and primers used for the restriction-free cloning are listed in Table S2.…”

Section: Methodsmentioning

confidence: 99%

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

Davidi

Moraïs

Artzi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca. The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.

show abstract

“…A restriction-free procedure was used to incorporate the Src kinase gene into pET28-TevH vector (27). The plasmids were transformed into E. coli BL21(DE3) cells.…”

Section: Expression and Purification Of Src Kinase And Caveolin 1 In mentioning

confidence: 99%

Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

Yosef¹,

Katz²,

Peleg³

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Much evidence points to a role of Na,K-ATPase in ouabaindependent signal transduction. Based on experiments with different cell lines and native tissue membranes, a current hypothesis postulates direct interactions between the Na,K-ATPase and Src kinase (non-receptor tyrosine kinase). Na,K-ATPase is proposed to bind Src kinase and inhibit its activity, whereas ouabain, the specific Na,K-ATPase inhibitor, binds and stabilizes the E2 conformation, thus exposing the Src kinase domain and its active site Tyr-418 for activation. Ouabain-dependent signaling is thought to be mediated within caveolae by a complex consisting of Na,K-ATPase, caveolin, and Src kinase. In the current work, we have looked for direct interactions utilizing purified recombinant Na,K-ATPase (human ␣1␤1FXYD1 or porcine ␣1D369N␤1FXYD1) and purified human Src kinase and human caveolin 1 or interactions between these proteins in native membrane vesicles isolated from rabbit kidney. By several independent criteria and techniques, no stable interactions were detected between Na,K-ATPase and purified Src kinase. Na,KATPase was found to be a substrate for Src kinase phosphorylation at Tyr-144. Clear evidence for a direct interaction between purified human Na,K-ATPase and human caveolin was obtained, albeit with a low molar stoichiometry (1:15-30 caveolin 1/Na,K-ATPase). In native renal membranes, a specific caveolin 1 4 -5 oligomer (95 kDa) was found to be in direct interaction with Na,K-ATPase. We inferred that a small fraction of the renal Na,K-ATPase molecules is in a ϳ1:1 complex with a caveolin 1 4 -5 oligomer. Thus, overall, whereas a direct caveolin 1/Na,K-ATPase interaction is confirmed, the lack of direct Src kinase/Na,K-ATPase binding requires reassessment of the mechanism of ouabain-dependent signaling.

show abstract

Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression

Cited by 224 publications

References 27 publications

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

Contact Info

Product

Resources

About