RF cloning: A restriction-free method for inserting target genes into plasmids

Ent, F. van den; Löwe, Jan

doi:10.1016/j.jbbm.2005.12.008

Cited by 563 publications

(418 citation statements)

References 9 publications

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“…Restriction free cloning was then used to produce a pET-41b expression vector containing PBP1b with a thrombin cleavable C-terminal His 8 tag (47).…”

Section: Methodsmentioning

confidence: 99%

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

King

Wasney

Nosella

et al. 2017

Journal of Biological Chemistry

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Edited by F. Peter GuengerichIn Escherichia coli, the peptidoglycan cell wall is synthesized by bifunctional penicillin-binding proteins such as PBP1b that have both transpeptidase and transglycosylase activities. The PBP1b transpeptidase domain is a major target of ␤-lactams, and therefore it is important to attain a detailed understanding of its inhibition. The peptidoglycan glycosyltransferase domain of PBP1b is also considered an excellent antibiotic target yet is not exploited by any clinically approved antibacterials. Herein, we adapt a pyrophosphate sensor assay to monitor PBP1b-catalyzed glycosyltransfer and present an improved crystallographic model for inhibition of the PBP1b glycosyltransferase domain by the potent substrate analog moenomycin. We elucidate the structure of a previously disordered region in the glycosyltransferase active site and discuss its implications with regards to peptidoglycan polymerization. Furthermore, we solve the crystal structures of E. coli PBP1b bound to multiple different ␤-lactams in the transpeptidase active site and complement these data with gel-based competition assays to provide a detailed structural understanding of its inhibition. Taken together, these biochemical and structural data allow us to propose new insights into inhibition of both enzymatic domains in PBP1b.

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“…Restriction free cloning was then used to produce a pET-41b expression vector containing PBP1b with a thrombin cleavable C-terminal His 8 tag (47).…”

Section: Methodsmentioning

confidence: 99%

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

King

Wasney

Nosella

et al. 2017

Journal of Biological Chemistry

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“…The DNA fragment encoding Thermus thermophilus ClpB ΔN (amino acids 143-854) was generated by PCR using Thermus thermophilus ClpB (7) as a template and subsequently cloned into a modified pET28b vector, with the thrombin cleavage site replaced by a six-histidine tag followed by a tobacco etch virus (TEV) protease cleavage site. The NTD domain of ClpB (amino acids 1-142) was prepared by a ligation-free cloning method (40,41) with residues 1-142 of ClpB PCR amplified with appropriate primers from a full-length ClpB vector and inserted into a pTWIN1 vector (NEB) immediately in front of the Mxe GyrA intein (an entire chitin binding domain, Ssp DnaB intein, and the MCS cassette was replaced by residues 1-142 of ClpB). An uncleavable hexahistidine tag was added at the N terminus of the construct by ligation of a duplex oligonucleotide containing five his codons (flanked by NdeI overhangs on both sides) into a NdeI-cleaved NTD plasmid.…”

Section: Methodsmentioning

confidence: 99%

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Rosenzweig

Farber

Vėlyvis

et al. 2015

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

114

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ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/ Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.T he heat shock protein ClpB (Escherichia coli) or Hsp100 (eukaryotes) is the main protein disaggregase in bacteria, yeast, plants, and mitochondria of all eukaryotic cells, and it is essential for cell survival during severe stress (1-4). Recovery of functional proteins from aggregates by ClpB requires the synergistic interaction with a second molecular chaperone, DnaK (1). Through its cochaperone, DnaJ, DnaK initially binds to the aggregates, leading to the exposure of peptide segments that can be recognized by ClpB (5, 6). DnaK then recruits ClpB to the site of aggregation through direct physical interaction (7, 8), transferring the aggregate to ClpB. Using the energy derived from ATP hydrolysis, ClpB unravels the aggregate by threading single polypeptide chains, one at a time, through the central pore of its hexameric ring (9). Once released from the aggregate, the unfolded polypeptides can either refold spontaneously or fold with the help of additional cellular chaperones.Like other Hsp100 proteins, ClpB forms a hexameric ring, with each protomer comprising an N-terminal domain (NTD) and two nucleotide binding domains (NBD1 and NBD2) separated by a unique regulatory coil-coil domain (10) essential for DnaK binding (7, 11) ( Fig. 1 A and B). Both NBDs contain Walk...

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“…Due to the presence of internal BamHI sites, Atg29 and the Atg13[CTD] were cloned using the restriction-free (RF) cloning method, 47 or site-directed mutagenesis followed by conventional restriction digestion cloning.…”

Section: Molecular Cloningmentioning

confidence: 99%

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

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(2015) Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms, Autophagy, 11:6, 891-905, DOI: 10.1080/15548627.2015 The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1 [CTD]) and Atg13 (Atg13 [CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1 [CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.

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RF cloning: A restriction-free method for inserting target genes into plasmids

Cited by 563 publications

References 9 publications

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

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