Folding and Purification of Insoluble (Inclusion Body) Proteins from <i>Escherichia coli</i>

Wingfield, Paul T.; Palmer, Ira; Liang, Shu‐Mei

doi:10.1002/0471140864.ps0605s00

Cited by 39 publications

(36 citation statements)

References 50 publications

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“…BLI can be used to determine whether a protein is present in a sample by means of specific antibodies. Purification of vimentin and vimentin fragments has been described elsewhere (Strelkov et al, 2001); expression and purification strategies can also be found elsewhere (Palmer & Wingfield, 2012; Wingfield, Palmer, & Liang, 2014). Purification of proteins tagged with His 6 or GST is much simpler than purification of untagged proteins (Palmer & Wingfield, 2012; Wingfield et al, 2014).…”

Section: Investigating Vimentin–protein Interactionsmentioning

confidence: 99%

Methods for Determining the Cellular Functions of Vimentin Intermediate Filaments

Ridge

Shumaker

Ahrends

et al. 2016

Methods in Enzymology

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The type III intermediate filament protein vimentin was once thought to function mainly as a static structural protein in the cytoskeleton of cells of mesenchymal origin. Now, however, vimentin is known to form a dynamic, flexible network that plays an important role in a number of signaling pathways. Here, we describe various methods that have been developed to investigate the cellular functions of the vimentin protein and intermediate filament network, including chemical disruption, photoactivation and photoconversion, biolayer interferometry, soluble bead binding assay, three-dimensional substrate experiments, collagen gel contraction, optical-tweezer active microrheology, and force spectrum microscopy. Using these techniques, the contributions of vimentin to essential cellular processes can be probed in ever further detail.

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Section: Investigating Vimentin–protein Interactionsmentioning

confidence: 99%

Methods for Determining the Cellular Functions of Vimentin Intermediate Filaments

Ridge

Shumaker

Ahrends

et al. 2016

Methods in Enzymology

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“…Using PCR with appropriate primers, an 83-residue segment from the N terminus of Pik1 consisting of residues , was tagged at its C terminus with an His 6 tract, inserted as an NcoI-XhoI fragment into the corresponding sites in the vector, pET23d, and expressed in E. coli strain BL21(DE3), as described above; in this construct, the Pik1-derived sequence is preceded by a 2-residue leader (Met-Ala-). Pik1-(110 -192)-(His) 6 was produced primarily in inclusion bodies, which were solubilized using 6 M guanidine hydrochloride (49) All NMR experiments were performed at 37°C on a Model DRX-500 or DRX-600 NMR spectrometer (Bruker Instruments, Billerica, MA) equipped with a four-channel interface and a triple-resonance probe with triple-axis pulsed field gradients as described before (35). The NMR spectra were processed and analyzed as described previously (50).…”

Section: S]met and [mentioning

confidence: 99%

Molecular Interactions of Yeast Frequenin (Frq1) with the Phosphatidylinositol 4-Kinase Isoform, Pik1

Huttner¹,

Strahl²,

Osawa³

et al. 2003

Journal of Biological Chemistry

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Recognition that phosphoinositides and inositol phosphates are key regulators of many processes in eukaryotic cells has brought increased attention to the enzymes that regulate the synthesis and turnover of these molecules (reviewed in Refs. 1-3). Of particular interest are the enzymes responsible for producing the various polyphosphoinositides situated on the cytosolic face of cellular membranes, which initiate several different signaling pathways by serving as highly specific recognition determinants for the selective recruitment of proteins to membranes (reviewed in Refs. 4 -7) and as the precursors for several intracellular second messengers (reviewed in Refs. 8 -10). The first committed step in the synthesis of the polyphosphoinositide, phosphatidylinositol 4,5-bisphosphate, is considered to be ATP-dependent phosphorylation of the hydrophilic myo-inositol head group of phosphatidylinositol (PtdIns) 1 at the D-4 position by PtdIns 4-kinase (ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC 2.7.1.67) (reviewed in Refs. 11-13) . The resulting product, PtdIns(4)P, can be phosphorylated on the D-5 position by PtdIns(4)P 5-kinase to generate PtdIns(4,5)P 2 , PtdIns(4,5)P 2 can be phosphorylated on the D-3 position by yet other lipid kinases, and the phosphoinositides so generated can be converted to other species by specific phosphatases and phospholipases (reviewed in Refs. 14 -17).The first PtdIns 4-kinase to be purified to homogeneity from any organism (18), and to have the corresponding gene cloned (19,20), was Pik1 from the yeast Saccharomyces cerevisiae. Thereafter, a second isoform, Stt4, which is the product of a discrete gene, was described (21). Absence of either Pik1 or Stt4 is lethal, and overproduction of each protein cannot compensate for absence of the other, indicating that these enzymes participate in distinct cellular processes and generate discrete pools of PtdIns(4)P that are essential for yeast cell viability. Indeed, subsequent work has shown that, together, Pik1 and Stt4 account for all of the PtdIns(4)P generated in the yeast cell (22) and that Pik1 is required for vesicular trafficking in the late secretory pathway (23, 24) and perhaps for cytokinesis (20), whereas Stt4 plays roles in cell wall integrity, maintenance of vacuole morphology, and aminophospholipid transport from the endoplasmic reticulum to the Golgi (25-27). The presence of Pik1-and Stt4-like isoforms is also conserved in metazoans (11,12,28).We have shown previously that Frq1, a small calcium-bind-

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“…) was MutS. However, a vast majority (ϳ80%) of the recombinant protein precipitated as inclusion bodies (30). This was corrected by reducing the isopropyl-1-thio-␤-D-galactopyranoside to 0.05-0.1 mM and lowering the growth temperature to 25°C.…”

Section: Purification Of Hexameric Histidine Recombinant Muts-mentioning

confidence: 99%

Role of MutS ATPase Activity in MutS,L-dependent Block of in Vitro Strand Transfer

Worth

Bader

Yang

et al. 1998

Journal of Biological Chemistry

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In addition to mismatch recognition, Escherichia coliMutS has an associated ATPase activity that is fundamental to repair. Hence, we have characterized two MutS mutant gene products to define the role of ATP hydrolysis in homeologous recombination. These mutants, denoted In addition to its role in replication fidelity, mismatch repair contributes to genome stability by controlling the level of recombination between closely related sequences (1-9). In Escherichia coli, MutS and MutL act to block recombinant yield when phenotypic selection arises in the vicinity of heteroallelic markers (10). Recent work from Zahrt and Maloy (11) provided more evidence implicating mismatch repair in recombination. Specifically, they showed that the efficiency of chromosomal gene transfer between Salmonella typhimurium and the closely related Salmonella typhi is low due to mismatch repair. Zahrt and Maloy (11) attributed this barrier of genetic exchange to DNA divergence and the ability of MMR to correct this event in favor of the recipient strand. Abdulkarim and Hughes (12) also showed that the mutHLSU genes increased the fidelity of exchange 1000-fold between the TufA and TufB translation factors, whose sequences share 99% sequence identity. Mismatch repair in this case ensures that the integrity of the genome is preserved by allowing these genes to evolve in concert.The first biochemical evidence to implicate mismatch repair in homologous recombination was provided by the in vitro RecA-catalyzed three-strand transfer reaction. E. coli MutS was shown to inhibit exchange between M13 and fd DNAs whose sequence heterology is ϳ3% at the nucleotide level (13). Since MutS was without effect on M13-M13 or fd-fd exchange, we attribute the block to the occurrence of mismatched base pairs in newly formed heteroduplex.MutL enhanced the MutS-dependent block of M13-fd exchange. Indeed, these proteins completely abolished full-length heteroduplex between these DNAs and is reminiscent of the proposed role of these activities in . MutL is believed to add to the MutS mismatch complex in an ATP-dependent fashion. Evidence for this stems from work by Grilley et al. (20), who showed the region of DNase I protection by MutS and ATP is extended by MutL. In the experiments described here, the role of ATP hydrolysis by MutS is examined on RecA-strand transfer. Previous work by Wu and Marinus (21) described dominant negative mutants of MutS from MNNG treatment and showed a large fraction of the isolates were missense mutations within the nucleotide binding domain (21). It is here that we have characterized two of these mutants, MutS501 and MutS506, and show that 1) mismatch binding is retained, 2) ATP hydrolysis is reduced, and 3) mismatch correction with MutS Ϫ extracts is not restored. These mutants also blocked RecA-catalyzed strand transfer between M13 and fd DNAs. That MutS501, and to a lesser extent MutS506, inhibited M13-fd and not M13-M13 exchange, suggests that mismatch recognition precludes RecA-dependent branch migration in regions of nonhomo...

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Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli

Cited by 39 publications

References 50 publications

Methods for Determining the Cellular Functions of Vimentin Intermediate Filaments

Methods for Determining the Cellular Functions of Vimentin Intermediate Filaments

Molecular Interactions of Yeast Frequenin (Frq1) with the Phosphatidylinositol 4-Kinase Isoform, Pik1

Role of MutS ATPase Activity in MutS,L-dependent Block of in Vitro Strand Transfer

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