Log In Sign up
The largest database of trusted experimental protocols

Pe sumo vector

Manufactured by LifeSensors
Visit Supplier
Sourced in United States

The PE-SUMO vector is a laboratory tool used for protein expression and purification. It contains a SUMO (Small Ubiquitin-like Modifier) tag that can be fused to a target protein, facilitating its expression and purification. The SUMO tag can be subsequently cleaved off, allowing for the isolation of the target protein.

Automatically generated - may contain errors

Lab products found in correlation

Talon metal affinity chromatography, by Takara Bio (2 mentions) Talon metal affinity resin, by Takara Bio (2 mentions) In fusion cloning, by Takara Bio (1 mentions) Gwiz vector, by Genlantis (1 mentions) Quikchange site directed mutagenesis method, by Agilent Technologies (1 mentions) Pcdna3 vector, by Thermo Fisher Scientific (1 mentions) Quickchange mutagenesis kit, by Agilent Technologies (1 mentions) Pcdna5 frt, by Thermo Fisher Scientific (1 mentions) Hiload 16 600 superdex 75 pg column, by GE Healthcare (1 mentions) Superdex 75 10 300 gl column, by GE Healthcare (1 mentions) Hitrap q hp anion exchange column, by GE Healthcare (1 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions) Hiload 16 60 superdex 200 column, by GE Healthcare (1 mentions) Hitrap sp hp, by GE Healthcare (1 mentions) Superdex 200 increase column, by GE Healthcare (1 mentions) Ni nta superflow, by Qiagen (1 mentions) Quikchange site directed mutagenesis, by Agilent Technologies (1 mentions) Histrap hp column, by GE Healthcare (1 mentions) Superdex 75 column, by GE Healthcare (1 mentions) Histrap column, by GE Healthcare (1 mentions)

Close spelling variants of this product

PE-SUMO (12 citations)

11 protocols using pe sumo vector

1

Mammalian Expression and Bacterial Purification of Engineered Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cmyc-R11.1.6, cmyc-YW1, HA-K-Ras G12D, HA-K-Ras WT, EGFP-R11.1.6, EGFP-YW1, and mApple-K-Ras G12D were cloned into the gWIZ vector (Genlantis) for mammalian expression using In-Fusion Cloning (Clontech) according to the manufacturer’s instructions. The sequence for R11.1.6 was PCR amplified from the yeast display vector (pCTCON2), digested with BsaI and XbaI, and cloned into BsaI digested pE-SUMO-vector (LifeSensors) for bacterial protein expression. To make the scrambled YW1 control and R11.1.6 binding interface alanine point mutants, the QuikChange site-directed mutagenesis method (Agilent) was used according to the manufacturer’s instructions. Transient transfections into HEK 293T cells were carried out using calcium phosphate. Briefly, DNA diluted in water was added to 2 M CaCl2, to which 2x HBS was added dropwise. The transfection mixture was added to plated cells and incubated for 8 hours, after which the transfection medium was replaced with complete medium. Unless indicated otherwise, the ratio of DNA transfected for K-Ras constructs to R11.1.6/YW1 constructs was 1:4.
Kauke M.J., Traxlmayr M.W., Parker J.A., Kiefer J.D., Knihtila R., McGee J., Verdine G., Mattos C, & Wittrup K.D. (2017). An engineered protein antagonist of K-Ras/B-Raf interaction. Scientific Reports, 7, 5831.
+ Open protocol
+ Expand
2

Purification of rcSso7d-based Binders

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Enriched PXDN-binders were sub-cloned into the pE-SUMO vector (Life-Sensors) and expressed in E. coli as fusion proteins comprising an N-terminal His6-tag, followed by small ubiquitin like-modifier (SUMO) and the respective binder, as described previously [35 (link)]. After purification of the His6-SUMO-binder fusion proteins using TALON metal affinity resin (Clontech), they were digested with SUMO protease 1, resulting in cleavage just before the N-terminus of the rcSso7d mutants. The digested products were again purified using TALON resin and the cleaved binders were collected from the flow through. A detailed description of the expression and purification of rcSso7d-based binders was published previously [35 (link)]. Plasmids encoding all engineered PXDN-specific binders will be made available to others.
Paumann-Page M., Kienzl N.F., Motwani J., Bathish B., Paton L.N., Magon N.J., Sevcnikar B., Furtmüller P.G., Traxlmayr M.W., Obinger C., Eccles M.R, & Winterbourn C.C. (2021). Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential. Redox Biology, 46, 102090.
+ Open protocol
+ Expand
3

Cloning and Mutagenesis of HIV-1 Vpr

Check if the same lab product or an alternative is used in the 5 most similar protocols
The Vpr with H78C mutation from HIV-1 (NL4-3 clone) was cloned into the pET43 (EMDBioscience) vector with a C-terminal His6 (link)-tag, modified to include a TEV protease site between the N-terminal soluble fusion NusA and the Vpr, as described previously29 (link). The WT Vpr was also cloned into pCDNA3 vector (Life Technologies) with an N-terminal HA tag. The cDNA encoding full-length UNG2 was cloned into the pCDNA3 vector with an N-terminal Myc tag, and the catalytic core domain (residues 83–304) was cloned into the pE-SUMO vector (LifeSensors). The N-terminally Myc-tagged DDB1 was cloned in to the pCDNA3 vector. The C-terminal region of DCAF1 (residues 1021–1400) was cloned into the pCDNA5/FRT (Life Technologies) with an N-terminal 3XFLAG-tag. Site-specific mutants of Vpr, UNG2 and DCAF1 were prepared using the Quickchange mutagenesis kit (Agilent Technologies). All other clones were described previously29 (link).
Wu Y., Zhou X., Barnes C.O., DeLucia M., Cohen A.E., Gronenborn A.M., Ahn J, & Calero G. (2016). The DDB1–DCAF1–Vpr–UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction. Nature structural & molecular biology, 23(10), 933-940.
+ Open protocol
+ Expand
4

Purification of p19 Protein Constructs

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
The PFO-based endosome-disrupting agent (C225.2/PFOT490A,L491V) was prepared as previously described (17 (link)). The p19, p19-E6, p19-E18 clones and SUMO-E18 were expressed from the pE-SUMO vector (LifeSensors) in Rosetta 2 (DE3) Escherichia coli (Novagen) and purified by Talon metal affinity chromatography (Clontech) following previously described methods (17 (link)). Following cleavage of the SUMO tag, the p19 constructs were purified by anion exchange chromatography (AEX) and size-exclusion chromatography (SEC). AEX was performed using a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences) with an increasing salt gradient (10–500 mM NaCl) in 20 mM Bis–Tris, pH 6.5. SEC was performed using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare Life Sciences) in PBS. Analytical SEC was performed using a Superdex 75 10/300 GL column (GE Healthcare Life Sciences) or Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) in PBS. Detailed methods for the expression and purification of p19 are provided in Supplementary Methods.
Yang N.J., Kauke M.J., Sun F., Yang L.F., Maass K.F., Traxlmayr M.W., Yu Y., Xu Y., Langer R.S., Anderson D.G, & Wittrup K.D. (2017). Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins. Nucleic Acids Research, 45(13), 7602-7614.
+ Open protocol
+ Expand
5

Production and Purification of AsCpf1 Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols
The gene encoding full-length AsCpf1 (residues 1–1307) was cloned between the NdeI and XhoI sites of the modified pE-SUMO vector (LifeSensors). The AsCpf1 protein was expressed at 20°C in Escherichia coli Rosetta2 (DE3) (Novagen), and was purified by chromatography on Ni-NTA Superflow (QIAGEN) and HiTrap SP HP (GE Healthcare) columns. The protein was incubated overnight at 4°C with TEV protease to remove the His6-SUMO-tag, and was then passed through the Ni-NTA column. The protein was further purified by chromatography on a HiLoad Superdex 200 16/60 column (GE Healthcare). The selenomethionine (SeMet)-labeled AsCpf1 protein was expressed in E. coli B834 (DE3) (Novagen), and purified using a similar protocol as that for the native protein. The crRNA was purchased from Gene Design. The target and non-target DNA strands were purchased from Sigma-Aldrich. The purified AsCpf1 protein was mixed with the crRNA, the target DNA strand, and the non-target DNA strand (molar ratio, 1:1.5:2.3:3.4), and then the reconstituted AsCpf1–crRNA–target DNA complex was purified by gel filtration chromatography on a Superdex 200 Increase column (GE Healthcare), in buffer consisting of 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and 1 mM DTT.
Yamano T., Nishimasu H., Zetsche B., Hirano H., Slaymaker I.M., Li Y., Fedorova I., Nakane T., Makarova K.S., Koonin E.V., Ishitani R., Zhang F, & Nureki O. (2016). Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell, 165(4), 949-962.
+ Open protocol
+ Expand
6

Generating Piwi and Siwi Mutants

Check if the same lab product or an alternative is used in the 5 most similar protocols
The slicer-Piwi mutant and the Siwi mutants were generated by inverse PCR, using pAcF-Piwi51 (link) and the FLAG-Siwi vector48 (link) as the templates, respectively. The gene encoding the Piwi PAZ domain (residues 262–374) was amplified by PCR using pAcF-Piwi51 (link) as the template, and then cloned into the pE-SUMO vector (LifeSensors). The sequences of the DNA oligos used for PCR are listed in Supplementary Table 1.
Yamaguchi S., Oe A., Nishida K.M., Yamashita K., Kajiya A., Hirano S., Matsumoto N., Dohmae N., Ishitani R., Saito K., Siomi H., Nishimasu H., Siomi M.C, & Nureki O. (2020). Crystal structure of Drosophila Piwi. Nature Communications, 11, 858.
+ Open protocol
+ Expand
7

Recombinant Protein Expression and Purification

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Fn3, E6rGel, and PFO variants were expressed using the pE-SUMO vector (LifeSensors, Malvern, PA) in Rosetta 2 (DE3) Escherichia coli (Novagen, San Diego, CA). Point mutations in PFO and E6rGel (C459A/T490A/L491V and Y74A/Y133A/E166K/R169Q, respectively) were introduced by QuikChange site-directed mutagenesis (Agilent, Santa Clara, CA). Briefly, bacterial cultures were grown to an OD600 of 2 in Terrific Broth (TB) and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 20 °C overnight. The proteins of interest were purified from sonicated pellets using Talon metal affinity chromatography (Clontech, Mountain View, CA) per the manufacturer’s protocol. Following an overnight digestion with SUMO protease at 4 °C, cleaved SUMO and SUMO protease were removed by Talon metal affinity chromatography. C225.2 was expressed and purified from HEK 293F cells as previously described.26 (link) All proteins were subjected to endotoxin removal as described below and stored in PBS.
Yang N.J., Liu D.V., Sklaviadis D., Gui D.Y., Vander Heiden M.G, & Wittrup K.D. (2015). Antibody-Mediated Neutralization of Perfringolysin O for Intracellular Protein Delivery. Molecular pharmaceutics, 12(6), 1992-2000.
+ Open protocol
+ Expand
8

Recombinant Chaperone and Tau Protein Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
The cDNAs of Apg2 (HSPH2), Hsc70 (HSPA8), DNAJB1 and DNAJA2 were obtained from Addgene and cloned into a pE-SUMO vector (LifeSensors, Malvern, USA). The deletion mutants DNAJA2ΔJD, DNAJA2ΔG/FR, DNAJA2ΔZFLR, DNAJA2ΔCD, and CD carrying a deletion from residue 1 to 74, 77 to 101, 141 to 209, 361 to 412, or 1–361, respectively, were cloned by fusing two PCR fragments corresponding to the upstream and downstream sequences of these protein segments. DNAJA2LQ was generated by restriction digest-based mutagenesis and DNAJA2QPN was produced by GeneScript (Rijswijk, Netherlands). All mutants were verified by sequencing. Recombinant chaperones containing a tag with 6xHis and SUMO fused to the N-terminus were expressed in BL21 Rosetta or BL21-CodonPlus (DE3)-RIPL cells and purified as described32 (link). Tau K18 C291A, C322A, P301L (K18 P301L) was cloned into a pNG2 vector, expressed in E. coli BL21 (DE3) cells and purified as described42 (link). Purification of human 6x His-tagged Hsc70Δlid was performed as reported36 (link), with an extra purification step. Briefly, after expression, the lysate was loaded onto a HisTrap column and the collected fractions were subjected to anion exchange on Q-Sepharose (pH 8.0), cation exchange on S-Sepharose (pH 6.5), and gel exclusion on Superdex 200.
Velasco-Carneros L., Cuéllar J., Dublang L., Santiago C., Maréchal J.D., Martín-Benito J., Maestro M., Fernández-Higuero J.Á., Orozco N., Moro F., Valpuesta J.M, & Muga A. (2023). The self-association equilibrium of DNAJA2 regulates its interaction with unfolded substrate proteins and with Hsc70. Nature Communications, 14, 5436.
+ Open protocol
+ Expand
9

Chaperone Protein Expression and Purification

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
All chaperones were expressed as fusion proteins containing an N-terminal 6x-His tag followed by small ubiquitin-like modifier (SUMO) and the chaperone using the pE-SUMO vector (LifeSensors, Malvern, PA). Proteins were expressed and purified as described previously using TALON metal affinity resin (Clonetech).24 (link) Chaperone sequences were amplified from alternate expression plasmids using primers specific to the N and C-termini with proper 5′ extensions to allow for cloning into the pE-SUMO vector (Fwd: CAGGTCTCAAGGT, Rev: GTTCTAGATTATTA). HSP90 was a gift from William Sessa (Addgene plasmid #22487),25 (link) pcDNA5/FRT/TO HSPA1A was a gift from Harm Kampinga (Addgene plasmid #19456),26 (link) and pNIC28-Bsa4-TF was a gift from Brian Smith (Addgene plasmid #61689).
Kelly R.L., Geoghegan J.C., Feldman J., Jain T., Kauke M., Le D., Zhao J, & Wittrup K.D. (2017). Chaperone proteins as single component reagents to assess antibody nonspecificity. mAbs, 9(7), 1036-1040.
+ Open protocol
+ Expand
10

Purification and storage of HIV-1 RNase H

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
The wild type RNH used for this experiment is the same construct used previously and was expressed and purified as reported 51 (link). In brief, cDNA encoding residues 427–560 of HIV-1 reverse transcriptase was expressed by inserting it into pE-SUMO vector (LifeSensors, Malvern, PA) with a six histidine tag (His6-) at the N-terminus of the SUMO-fusion construct. The protein was expressed in E. Coli Rosetta 2 (DE3) cells using IPTG for induction. Soluble RNH was extracted from the cell lysate and purified using HisTrap HP columns (GE Healthcare, Piscataway, NJ) and gel filtration on a Superdex75 column (GE Healthcare, Piscataway, NJ). After removal of the N-terminal His6-SUMO fusion by digestion with ULP1 enzyme, the RNH was separated from His-tagged proteins using a HisTrap column (GE Healthcare, Piscataway, NJ). Purified RNH was stored at −80 °C with a buffer containing 25 mM Sodium Phosphate, 100 mM NaCl and 0.02% NaN3, at pH 7.0. The buffers for NMR experiments were exchanged as described below.
Karki I., Christen M.T., Spiriti J., Slack R.L., Oda M., Kanaori K., Zuckerman D.M, & Ishima R. (2016). Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: a Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain. The journal of physical chemistry. B, 120(49), 12420-12431.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!