For H6-small ubiquitin-related modifier (SUMO), LB Amp/Cam + 0.1% glucose (10 mL) was inoculated with E. coli Rosetta transformed with pET-SUMO (Thermo Fischer Scientific) and incubated at 37°C ON. A 4 mL-fraction of the preculture was added to 400 mL LB Amp/Cam + 0.1% glucose that was incubated at 37°C until OD600nm = 0.6. Expression of SUMO was induced by the addition of 0.1 mM IPTG. The culture was further incubated at 37°C for 2.5 h and centrifuged at 3000 × g for 15 min at 4°C. The pellet was resuspended in 10 mL of lysis buffer 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 20 mM imidazole, 1/100 protease inhibitor cocktail set III (Calbiochem). The bacteria in suspension were lysed with several 30 s-long sonication pulses using the apparatus Labsonic 2000 (Braun). Lysates were centrifuged at 20,000 × g at 4°C. The supernatant was filtrated through the filter Millex GV, 0.22 uM, and loaded onto the column HisTrap 1 mL (GE Healthcare). H6-SUMO was eluted with the lysis buffer supplemented with 0.5 M imidazole, dialyzed against PBS, flash frozen in liquid nitrogen and kept at -80°C.
Pet sumo
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The PET-SUMO is a laboratory instrument designed for the purification of recombinant proteins using the SUMO (Small Ubiquitin-like Modifier) fusion system. The core function of the PET-SUMO is to facilitate the efficient removal of the SUMO tag from the target protein, enabling the isolation of the purified protein.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (4 mentions)
Hitrap sp hp, by GE Healthcare (3 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (2 mentions)
Sybr gold, by Thermo Fisher Scientific (2 mentions)
Histrap hp nickel column, by GE Healthcare (2 mentions)
Hiload 16 60 superdex 75 column, by GE Healthcare (2 mentions)
In fusion hd cloning kit, by Takara Bio (2 mentions)
Petsumo, by Takara Bio (2 mentions)
Accuprime pfx dna polymerase, by Thermo Fisher Scientific (2 mentions)
Sumo protease, by Thermo Fisher Scientific (2 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Kta fplc system, by GE Healthcare (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pet15b vector, by Merck Group (1 mentions)
His60 ni superflow resin, by Takara Bio (1 mentions)
Biologic lp system, by Bio-Rad (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Quikchange site directed mutagenesis, by Thermo Fisher Scientific (1 mentions)
Pmsp1d1 plasmid, by Addgene (1 mentions)
26 protocols using pet sumo
1
Recombinant SUMO Protein Expression
2
Recombinant Expression and Purification of rRrp2
3
Plasmid Acquisition and Characterization
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The Zero Blunt PCR vector was obtained from Thermo Fisher Scientific, the pET15b vector was obtained from EMD Millipore (Billerica, MA, USA), the pHis-Trx vector was kindly provided by Professor Andrea Brancaccio from the Consiglio Nazionale delle Richerche, and pET-SUMO was obtained from Thermo Fisher Scientific. Other chemical reagents were of analytical grade.
4
Purification of LysM Proteins
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Coding sequences of mature proteins without signal peptide were amplified with primers listed in Table S2 and cloned into expression vector pETSUMO (Thermo Fisher Scientific, Massachusetts, USA). Correctness of the resulting constructs pETSUMO-ChElp2 and pETSUMO-Vd2LysM were confirmed by DNA sequencing and introduced into E. coli strains BL21 and Origami, respectively. Both proteins were produced at 28°C with 0.2 mM IPTG. Cell culture was pelleted by centrifugation at 4000 g for 40 min at 4°C, and the pellet was resuspended in 20 mL lysis buffer (Table S2), shaken at 4°C for at least two hours and centrifuged at 10,000 g for 1 h. The supernatant was collected and purified using His60 Ni Superflow resin (TaKaRa, California, USA) on a BioLogic LP system (Bio-Rad, California, USA). The resulting protein was dialyzed 3 L of 20 mM Tris, 150 mM NaCl, 5 % glycerol, pH 8.0 while 5 µL of cleavage protein ULP1 was added into the dialysis membrane to cleave-off the 6×His-SUMO tag. Next day, protein solution was collected and subjected to purification using His60 Ni Superflow resin (TaKaRa, California, USA) to remove 6×His-SUMO tag from the protein preparations. Eventually, LysM proteins were dissolved in 20 mM Tris, 150 mM NaCl, 5% glycerol, pH 8.0 and concentrated to a high concentration.
5
Molecular Tools for Shank2 and GKAP
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A construct coding for mCherry-tagged human Shank2a was obtained from Dr. Simone Berkel (Heidelberg, Germany) [29] . The Shank2a cDNA was cloned into the pHAGE-GFP vector for transfecting neurons. A rat cDNA for CortBP1 (Shank2b), donated by Dr. J. Thomas Parsons (University of Virginia) was cloned into pEGFP-C2, leading to expression of N-terminally GFP-tagged Shank2b. cDNA coding for GKAP was obtained from Prof. Stefan Kindler (UKE, Hamburg). Mutations were introduced using the QuikChange II site-directed mutagenesis kit (Agilent). cDNA coding for the 50 C-terminal residues of GKAP was amplified by PCR with appropriate primers and subcloned into BamHI/EcoRI sites of pGEX-4T2, allowing for expression as a GST fusion. For expression of the Shank2-SAM domain, cDNA coding for residues T1780-R1849 was cloned into pET-SUMO (Thermo-Fisher), allowing for expression of a SUMO-SAM fusion with an N-terminal His 6 -tag. A cDNA-fragment coding for SH3 to PDZ domains of Shank2 (R520-D727) was cloned into pET-SUMO. shRNA constructs were generated in pSuper based on constructs used by Berkel et al. [29] carrying target sequences GGATAAACCGGAAGAGAT (shShank2#1) and GGAATTGAGCAAAGAGATT (shShank2#2). A construct coding for rat Homer1b with an N-terminal GFP-tag in pEGFP-C1 was described [30] .
6
Cloning and Manipulation of Bacillus subtilis Genes
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The ccpC, darB, rel, sasA, and sasB alleles were amplified using chromosomal DNA of B. subtilis 168 as the template and appropriate oligonucleotides that attached specific restriction sites to the fragment. Those were: KpnI and BamHI for cloning rel in pGP17251 (link), BamHI and SalI for cloning rel in pWH84452 (link), XbaI and KpnI for cloning all genes in the BACTH vectors53 (link), BamHI and KpnI sites for cloning rel into pGP88854 (link) for genomic integration. The truncated rel variants were constructed as follows: rel-SYN-RRM contained aa 168–734, rel-HYD-SYN contained aa 1–391. For the overexpression of DarB, darB was amplified using chromosomal DNA of B. subtilis 168 as the template and appropriate nucleotides that attached BsaI and XhoI restriction sites to the fragments and cloned between the BsaI and XhoI sites of the expression vector pET-SUMO (Invitrogen, Germany). The resulting plasmid was pGP2972. All plasmids and oligonucleotides are listed in Supplementary Tables 2 , 3 , respectively.
7
Recombinant Anthrax Protein Production
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Recombinant wild-type (WT) PA was expressed in the periplasm of Escherichia coli BL21 (DE3) and purified by anion exchange chromatography [40 (link)] after activation of PA with trypsin [41 (link)]. QuikChange site-directed mutagenesis (Stratagene) was used to introduce mutations into the plasmid (pET SUMO (Invitrogen)) encoding a truncated recombinant portion of lethal factor. LFN E126C and was expressed as His6-SUMO-LFN, which was later cleaved by SUMO (small ubiquitin-related modifier) protease, revealing the native LFN E126C N-terminus [41 (link)]. Membrane scaffold protein 1D1 (MSP1D1) was expressed from the pMSP1D1 plasmid (AddGene) with an N-terminal His-tag and was purified by immobilized Ni-NTA affinity chromatography as previously described [42 (link)].
8
Purification and Characterization of TtAgo
9
Heterologous Expression of Vd2LysM Protein
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The coding sequence of Vd2LysM was amplified from V. dahliae cDNA and cloned into the pETSUMO (Invitrogen) expression vector according to the manufacturer's instructions prior to E. coli Origami (DE3) transformation. A single transformant was selected and grown in Luria broth medium until an optical density at 600 nm (OD600) of 0.9 was reached. Heterologous production of Vd2LysM was induced with 1 mm Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 26°C during ∼20 h. Cell pellets were lysed using lysozyme from chicken egg (Sigma, St Louis, MO, USA) and Vd2LysM was purified from the soluble protein fraction using an Ni2+‐NTA Superflow column (Qiagen). Purified protein was dialysed against 200 mm NaCl and concentrated over Amicon ultracentrifugal filter units (Molecular weight cut‐off (MWCO) = 3 kDa; Millipore, Billerica, MA, USA).
10
Heterologous Expression and Purification of FtsA
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To express the protein FtsA of B. cenocepacia J2315, the gene ftsA (BCAL3458) was amplified by PCR, using the primers ftsASUMOfor and ftsASUMOrev (Table S2 ), with genomic DNA as template. The fragment obtained was purified and inserted into the pETSUMO (Invitrogen) plasmid, using the In-fusion HD Cloning Kit protocol (Takara), and then the pETSUMO-FtsA (Table S3 ) was transformed into E. coli BL21(DE3) competent cells by electroporation. The culture was grown in LB supplemented with Kanamycin and the protein production was induced with 0.5 mM of IPTG overnight at 20 °C. The cells were harvested by centrifugation, resuspended in buffer E (50 mM Tris-HCl pH = 7.5, 300 mM NaCl, 5 mM MgCl2, glycerol 5%) containing 1 mM of PMSF and lysed by sonication. The lysate was clarified by centrifugation at 50,000× g for 30 min and the supernatant was applied on a HisTrap HP nickel column (1 mL, GE Healthcare), eluting the protein with 250 mM imidazole in buffer E. Then, the purified protein was dialyzed overnight against buffer B and the SUMO protease was used to remove the SUMO protein. A further purification step was carried out by size exclusion chromatography, using a HiLoad 16/60 Superdex-75 column (GE Healthcare) in buffer D, obtaining the purified FtsA that was concentrated to 5 mg/mL and stored at −80 °C.
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