High-Yield Expression and Purification of Recombinant Proteins in Bacteria: A Versatile Vector for Glutathione S-Transferase Fusion Proteins Containing Two Protease Cleavage Sites

Sehgal, Bernd U.; Dunn, Rebecca; Hicke, Linda A; Godwin, Hilary

doi:10.1006/abio.2000.4569

Cited by 7 publications

(7 citation statements)

References 7 publications

(1 reference statement)

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“…Construction of GST-C2 (encoding Rsp5 amino acids 1–142; LHP1663), rsp5 C2Δ (LHP510), rsp5 ww1 (LHP730), rsp5 ww2 (LHP845), rsp5 ww3 (LHP846), rsp5 ww1,2,3 (LHP952), and STE2-GFP (LHP1921) plasmids has been described previously ( Sehgal et al, 2000 ; Dunn and Hicke, 2001 ; Shih et al, 2002 ). p GST-C2B was a gift from H. Godwin (Northwestern University, Evanston, IL).…”

Section: Methodsmentioning

confidence: 99%

“…p GFP-C2 (LHP1527) was made by introducing into p GFP-RSP5 two in-frame stop codons after the Ser140 codon of RSP5 by the Quikchange method (Stratagene). The GST-3 × WW plasmid (LHP703) was constructed by subcloning all three WW domains of Rsp5, generated by PCR amplification of RSP5 with oligonucleotides containing nontemplated EcoRI (5′) or XhoI (3′) sites, into EcoRI- and XhoI-digested pGEX-PKT ( Sehgal et al, 2000 ). The 5K→Q mutations (K44Q, K45Q, K75Q, K77Q, and K78Q) were introduced by sequential site-directed mutagenesis of p GST - C2 , pNotI- RSP5, p GFP-RSP5 , and p GFP-C2 to generate p GST - C2 5K →Q (LHP1709), p RSP5 5K →Q (LHP1710), p GFP-RSP5 5K →Q (LHP1711), and p GFP-C2 5K →Q (LHP1713), respectively.…”

Section: Methodsmentioning

confidence: 99%

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The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo

Dunn

Klos

Adler

et al. 2004

The Journal of Cell Biology

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139

152

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Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin. Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole. A mutant lacking the Rsp5 C2 domain is unable to ubiquitinate or sort biosynthetic cargo into MVE vesicles, whereas endocytic cargo is ubiquitinated and sorted efficiently. The C2 domain binds specifically to phosphoinositides in vitro and is sufficient for localization to membranes in intact cells. Mutation of a lysine-rich patch on the surface of the C2 domain abolishes membrane interaction and disrupts sorting of biosynthetic cargo. Translational fusion of ubiquitin to a biosynthetic cargo protein alleviates the requirement for the C2 domain in its MVE sorting. These results demonstrate that the C2 domain specifies Rsp5-dependent ubiquitination of endosomal cargo and suggest that Rsp5 function is regulated by membrane phosphoinositides.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo

Dunn

Klos

Adler

et al. 2004

The Journal of Cell Biology

Self Cite

139

152

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“…A plasmid encoding GST fused to the three WW domains of RSP5 (pGST-3ϫWW, LHP703) was generated by PCR amplification of the WW domains (codons 228 -430) using a 5Ј EcoRI site-containing oligonucleotide and a 3Ј XhoI site-containing oligonucleotide. The PCR product was digested and ligated into EcoRI-and XhoI-digested pGEX-PKT (37).…”

Section: Methodsmentioning

confidence: 99%

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

Stamenova¹,

Dunn²,

Adler

et al. 2004

Journal of Biological Chemistry

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Sla1 and Rvs167 are yeast proteins required for receptor internalization and organization of the actin cytoskeleton. Here we provide evidence that Sla1 and Rvs167 are orthologues of the mammalian CIN85 and endophilin proteins, respectively, which are required for ligand-stimulated growth factor receptor internalization. Sla1 is similar in domain structure to CIN85 and binds directly to the endophilin-like Rvs167. Akin to CIN85, Sla1 interacts with synaptojanins and a ubiquitin ligase that regulates endocytosis. This ubiquitin ligase, Rsp5, binds directly to both Sla1 and Rvs167. The interaction between Rsp5 and Rvs167 is mediated through Rsp5 WW domains and PXY motifs in the central Gly-Pro-Ala-rich domain of Rvs167. Rvs167 PXY motifs are required for Rsp5-dependent monoubiquitination of Rvs167 on Lys 481 in the Src homology 3 (SH3) domain. Mutation of Lys 481 3 Arg causes cells to grow slowly on medium containing 1 M NaCl, although this phenotype is not due to the defect in ubiquitination caused by the K481R mutation. We propose that Rsp5 interaction with Sla1-Rvs167 promotes Rvs167 ubiquitination and regulates activity of this protein complex. Rvs167 ubiquitination is not required for general function of Rvs167, but may control specific Rvs167 SH3 domain-protein interactions or negatively regulate SH3 domain activity.

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“…The redesigned proteins (i.e., CD2D1-Ca1, CD2D1-Ca9, and CD2D1-Ca18) were constructed using standard site-directed mutagenesis techniques 50 using the first domain of the wild-type CD2 gene cloned into vector pGEX-PKT, which includes an N-terminal fusion to a poly-Gly linker and glutathione S-transferase (GST) protein (plasmid pGEX-PKT was a gift from Dr. H. Godwin). 51 The GST fusion protein were purified from bacterial lysates by affinity chromatography using immobilized glutathione agarose matrix, which can capture the GST moiety. Subsequently, the impurities are removed by washing, and the protein of interest is cleaved and released using a site-specific protease.…”

Section: Methodsmentioning

confidence: 99%

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

2008

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One of the many challenging tasks of protein design is the introduction of a completely new function into an existing protein scaffold. In this study, we introduce a new computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. This is accomplished by introducing a two-level procedure where we first identify where are the most appropriate locations to graft the new binding pocket into the protein fold by minimizing the departure from a set of geometric restraints using mixed-integer linear optimization. On identifying the suitable locations that can accommodate the new binding pocket, CHARMM energy calculations are employed to identify what mutations in the neighboring residues, if any, are needed to ensure that the minimum energy conformation of the binding pocket conserves the desired geometry. This computational framework is benchmarked against the results available in the literature for engineering a copper binding site into thioredoxin protein. Subsequently, OptGraft is used to guide the transfer of a calcium-binding pocket from thermitase protein (PDB: 1thm) into the first domain of CD2 protein (PDB:1hng). Experimental characterization of three de novo redesigned proteins with grafted calcium-binding centers demonstrated that they all exhibit high affinities for terbium (K d $ 22, 38, and 55 lM) and can selectively bind calcium over magnesium.

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High-Yield Expression and Purification of Recombinant Proteins in Bacteria: A Versatile Vector for Glutathione S-Transferase Fusion Proteins Containing Two Protease Cleavage Sites

Cited by 7 publications

References 7 publications

The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo

The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

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