Primers for PCR were designed from BmSOD1 and BmSOD2 cDNA sequences obtained from GenBank and RefSeq (BmSOD1, Accession no. AB179561; BmSOD2, AB190802). Total RNA was extracted from the fat body of day 3 fifth instar larvae using an RNeasy mini kit (Qiagen). DNase-treated total RNA was processed for cDNA synthesis using oligo(dT)12–18 primers and SuperScript II reverse transcriptase (Invitrogen). The ORFs of BmSOD1 and BmSOD2 were amplified by PCR using PfuTurbo DNA polymerase (Agilent Technologies) with the following primers: BmSOD1, 5′-CCCGCCAAAGCAGTTTGCGTACTTC-3′ and 5′-TTAAATCTTGGCCAAGCCAATGACT-3′; BmSOD2, 5′-TTAATGTCACAAAGGATTGGATCA-3′ and 5′-TCACTTGAGCGCTTTTTCATATCT-3′. Products were cloned into prokaryotic expression vector pTrcHis-TOPO using a TOPO TA cloning kit (Invitrogen) and were expressed in Escherichia coli XL-1 blue strain as fusion proteins with N-terminal Xpress tags. The nucleotide sequence was confirmed by DNA sequencing. Recombinant BmSOD1 and BmSOD2 expressed in E. coli were purified with HIS-Select spin columns (Sigma, St. Louis, MO) according to supplier instructions.
His select spin columns
Manufactured by Merck Group
Sourced in United States
HIS-select spin columns are a type of affinity chromatography column designed to purify proteins with a histidine-tag. The columns contain a resin that binds to the histidine-tag, allowing the target protein to be separated from other components in the sample. The core function of these columns is to facilitate the purification of histidine-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pfu turbo dna polymerase, by Agilent Technologies (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nupage mops running buffer, by Thermo Fisher Scientific (1 mentions)
Celllytic b reagent, by Merck Group (1 mentions)
Nap 5 sephadex g 25 column, by GE Healthcare (1 mentions)
6 protocols using his select spin columns
1
Cloning and Expression of BmSOD1 and BmSOD2 in E. coli
2
Probing Protein-Protein Interactions
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To test the effect of replacing key residues that the structure implied would be important for complex formation, we generated RsbN91(L70R) and RsbN91(F14R-L50R) mutants and assessed their abilities to interact with BldN σ2 and BldN σ4, respectively. Specifically, RsbN91(L70R) was co-expressed with BldN σ2 and RsbN91(F14R-L50R) was co-expressed with BldN σ4 (Supplementary Table S1 ). Proteins were co-expressed in E. coli as above but instead purified using HIS-select spin columns (Sigma H7787) and run on 16% Tricine gels (47 (link)) for visualization with Coomassie Brilliant Blue.
To probe the BldN σ4–RsbN α3 interaction, a co-expression system in which the wild-type (WT) full length BldN and a fusion construct consisting of the maltose binding protein (MBP) connected via a linker (SHSGGGSGGGS) to RsbN α3 (herein termed MBP-RsbN α3) were encoded in pETDuet-1 (Supplementary Table S1 ). For protein expression, the plasmid was transformed into C41(DE3) cells and grown in LB to an OD600 of 0.6 and induced with 1 mM IPTG for 3 h at 28°C. The cells were suspended in buffer A, disrupted with a microfluidizer and the lysate mixed with cobalt-nitrilotriacetic acid resin. Protein containing fractions were then applied (at a concentration of 10 μM) to a HiLoad 26/600 Superdex 75 column in buffer A.
To probe the BldN σ4–RsbN α3 interaction, a co-expression system in which the wild-type (WT) full length BldN and a fusion construct consisting of the maltose binding protein (MBP) connected via a linker (SHSGGGSGGGS) to RsbN α3 (herein termed MBP-RsbN α3) were encoded in pETDuet-1 (
3
Purification of RsiG–WhiG Complexes
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E. coli carrying pCOLADuet-1 RsiG–WhiG coexpression constructs was grown at 37 °C in 50 mL LB medium with antibiotics to an OD600 of 0.45, then induced with 1 mM isopropyl β-d -thiogalactopyranoside (IPTG) at 37 °C overnight. Cells were harvested by centrifugation, resuspended in 1 mL Equil Buffer (Sigma-Aldrich) and lysed by sonication. Cell debris was removed by centrifugation and RsiG–WhiG complexes were purified on HIS-Select Spin columns (Sigma-Aldrich).
4
Purification of Recombinant Proteins from E. coli
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Overnight E. coli cultures grown at 37 °C were diluted 1:50 with LB medium, grown to OD600 of 0.5–0.8, induced by adding isopropyl-β-D-thiogalactoside (IPTG) to 1 mM and incubated for further 2 h. Large scale preparation was carried out using 100 ml of induced cultures. Bacteria were pelleted by centrifugation and resuspended in CellLytic B reagent (Sigma). After centrifugation the soluble fraction of the lysate was used for protein purification using HisSelect™ Spin Columns (Sigma) according to manufacturer’s protocol. The fractions were analyzed on 12 % NuPAGE™ Novex Bis-Tris SDS–PAGE gels.
The gels were run using NuPAGE™ MOPS running buffer (Invitrogen) at 200 V for 1 h and stained with Coomassie Blue.
The gels were run using NuPAGE™ MOPS running buffer (Invitrogen) at 200 V for 1 h and stained with Coomassie Blue.
5
Cloning, Expression, and Purification of Cbl-b and Tyro3 Kinases
6
Engineered CR2-sFlt 1-GFP Fusion Protein
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To generate the CR2-sFlt 1-green fluorescent protein (GFP) fusion protein, we cloned complementary DNA (cDNA) sequences consisting of four N-terminal SCRs of human CR2 (NM_001006658.2) linked to human sFlt 1-His tag (RefSeq: NM_001159920.1) with a signal peptide fragment at the N-terminus of sFlt 1 sequence into eukaryotic green fluorescent protein recombinant vector (pEGFP-N2) (Figure 1 ). The signal peptide fragment within the CR2-sFlt 1-GFP fusion protein facilitated CR2-sFlt 1-GFP to localize outside the cells. The polyhistidine (His)-tags were used for the affinity purification of genetically modified proteins. The final plasmid constructs were transfected into HeLa cells to express the CR2-sFlt 1-GFP fusion protein, and the GFP gene was used as a reporter and directly tracked the fusion expression under a fluorescence microscope. HIS-Select Spin Columns (H7787; Sigma-Aldrich Co., St Louis, MO, USA) were employed to purify the CR2-sFlt 1-GFP fusion protein in accordance with the manufacturer’s instructions, and the concentration of the purification protein was determined using the bicinchoninic acid (BCA) Protein Assay Kit (23225; Thermo Fisher Scientific, Waltham, MA, USA).
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