Individual human Vps26, Vps29, and GST-tagged Vps35 subunits were expressed individually in E. coli, and the assembled trimeric retromer complex was immobilized on GSH resin via the GST-tag on Vps35 as described [45 (link)]. Maltose binding protein (MBP)-L2–6His fusion proteins containing a C-terminal segment from wild-type or mutant HPV16 L2 (amino acids 434–457) were expressed in bacteria and purified using the AKTA-Prime plus FPLC system equipped with a His-trap column. The sequence appended to the C-terminus of MBP in the pMal-C2 expression vector (New England Biolabs) was GSASPQYTIIADAGDFYLHPSYYMLRK HHHHHHC (L2 sequence underlined). Purified proteins were exchanged into 20mM Hepes pH 8, 50mM NaCl and quantified by bicinchoninic acid assay. Five or 10 μM of each fusion protein was incubated with assembled retromer trimer immobilized on GSH resin for two hours at 4°C in 20mM HEPES pH 8.0, 50mM NaCl, 5mM MgCl2, 1mM DTT, and 0.1% Triton X-100. Beads were washed twice in HEPES buffer, suspended in SDS loading buffer, boiled, and subjected to SDS-PAGE and anti-His immunoblotting. Bands corresponding to the MBP-L2-His constructs were quantified by Image Lab.
Pmal c2 expression vector
Manufactured by New England Biolabs
Sourced in Japan
The PMAL-c2 expression vector is a plasmid used for the expression of recombinant proteins in Escherichia coli. It contains the malE gene, which encodes the maltose-binding protein (MBP), and allows for the expression of target proteins as fusions with MBP.
Automatically generated - may contain errors
7 protocols using pmal c2 expression vector
1
Retromer Complex Binding Assay
2
Mutagenesis and Purification of AHA2 H+-ATPase
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A cDNA of AHA2 H+-ATPase (Pardo and Serrano, 1989 (link)) was amplified with primers AHA2Ct-F and AHA2Ct-R (Supplementary Table S1 at JXB online) resulting in a fragment of 323bp encoding the last 106 aa of the protein, from R842 to V948. It was cloned into the pMAL-c2 expression vector (New England Biolabs, MA, USA) behind and in frame with maltose-binding protein (MBP). This construct was utilized as a template to generate point mutations by overlap-extension PCR (Patel et al. 2009 ). The primers for the mutations S899P, S904L, S931F, and T924A are shown in Supplementary Table S1 . As indicated, all were designed to introduce a new restriction site. To obtain the double mutant S931F T924A, the plasmid with the S931F mutation was used as template to introduce the second mutation. All mutations were verified by sequencing. The recombinant fusion proteins (MBP–CtAHA2) were purified by amylose affinity chromatography following the instructions of the vector manufacturer.
The purification of recombinant His-OST1/SnRK2.6 was as described by Fujii et al. (2009) (link).
The purification of recombinant His-OST1/SnRK2.6 was as described by Fujii et al. (2009) (link).
3
SARS-CoV-2 Main Protease Purification
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The MBP-tagged main protease containing SARS-CoV-2 Mpro (GenBank Accession No. YP_009725301; Met1–Gln306) was constructed using pMAL-c2 expression vector (New England Biolabs Japan Inc.). The gene encoding SARS-CoV-2 Mpro with E. coli codon usage was synthesized by Eurofins Genomics (Tokyo, Japan). The fusion protein was purified by amylose-conjugated affinity chromatography. MBP-cleaved Mpro was prepared from MBP-tagged Mpro by Factor Xa protease digestion. The prepared Mpro showed a single band (MBP-tagged Mpro, 77.5 kDa; MBP-cleaved Mpro, 34.7 kDa) (Supplemental Fig. 1). The concentration of each protein was estimated by bicinchoninic acid protein assay.
4
Cloning and Mutagenesis of hDGKδ
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Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding hDGKδ (accession No.NM_152879 ) plus a C-terminal PA tag (GVAMPGAEDDVV) [20 (link)] was subcloned into the pMAL-c2 expression vector (New England Biolabs Inc., Beverly, MA) using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan). The PA tag is recognized by an anti-PA tag mAb (NZ-1) [21 (link)]. The resulting construct was named pMAL-c2-hDGKδ-PA. The deletion mutants of hDGKδ DNA were amplified via polymerase chain reaction, and subcloned into the pMAL-c2 with a PA tag using the In-Fusion HD Cloning Kit. The substitution of hDGKδ amino acids with alanine on dN610 of hDGKδ was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). These constructs were also verified by direct DNA sequencing.
5
Recombinant SlWRKY3 Protein Expression
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SlWRKY3 was cloned into the pMAL-c2 expression vector (New England Biolabs) between BamHI and PstI restriction sites, allowing in frame fusion of SlWRKY3 with the maltose binding protein (MBP) coding sequence. Construct was introduced into the Escherichia coli BL21 strain. SlWRKY3 expression was induced with 1 mM isopropyl β-D -1-thiogalactopyranoside, during 5 h at 37°C. Protein binding array experiments were conducted as described in Hichri et al. (2014) (link).
6
Cloning and Expression of Arabidopsis Spliceosome Proteins
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AtMago and AtMagoΔC cDNAs were first amplified from qrt;HAP1/hap1-2 mutant cDNAs using primers AtMagoFBamHI and AtMagoRHindIII (AtMago) or AtMagoFBamHI and AtMagoΔCRHindIII (AtMagoΔC), respectively. The PCR products were cloned into pCRII-TOPO vector (Invitrogen). Then AtMago and AtMagoΔC were isolated by Hind III and BamH I and cloned into pMAL-c2 expression vector (NEB). AtY14 and AteIF4AIII were amplified from qrt cDNAs using AtY14FNdeI and AtY14RSalI primer pair and AteIF4AIIIFNdeI and AteIF4AIIIRSalI primer pair, respectively. The PCR products were cloned into pCRII-TOPO vector. Then AtY14 and AteIF4AIII were isolated by Nde I and Sal I. The released fragments were subsequently cloned into pET16b expression vector (Novagen). GFP, AtMago and AtMagoΔC were amplified separately, fused together by overlapping PCR to yield GFP-AtMago and GFP-AtMagoΔC. Then GFP-AtMago and GFP-AtMagoΔC were cloned between a 35S promoter and a NOS terminator on an expression vector described before [24 (link)]. CFP and DCP1 cDNA were also amplified separately, fused together by overlapping PCR to yield CFP-DCP1 fusion gene. Then CFP-DCP1 was cloned between a 35S promoter and a NOS terminator as described before [24 (link)]. All sequences were confirmed by sequencing at Genewiz Inc. All primer sequences were listed in S1 Table .
7
Production and Purification of SARS-CoV-2 Main Protease
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The MBP-tagged main protease containing SARS-CoV-2 M pro (GenBank Accession No.
YP_009725301; Met1-Gln306) was constructed using pMAL-c2 expression vector (New England Biolabs Japan Inc.). The gene encoding SARS-CoV-2 M pro with E. coli codon usage was synthesized by Eurofins Genomics (Tokyo, Japan). The fusion protein was purified by amylose-conjugated affinity chromatography. MBP-cleaved M pro was prepared from MBP-tagged M pro by Factor Xa protease digestion. The prepared M pro showed a single band (MBP-tagged M pro , 77.5 kDa; MBP-cleaved M pro , 34.7 kDa) (Supplemental Fig. 1). The concentration of each protein was estimated by bicinchoninic acid protein assay.
YP_009725301; Met1-Gln306) was constructed using pMAL-c2 expression vector (New England Biolabs Japan Inc.). The gene encoding SARS-CoV-2 M pro with E. coli codon usage was synthesized by Eurofins Genomics (Tokyo, Japan). The fusion protein was purified by amylose-conjugated affinity chromatography. MBP-cleaved M pro was prepared from MBP-tagged M pro by Factor Xa protease digestion. The prepared M pro showed a single band (MBP-tagged M pro , 77.5 kDa; MBP-cleaved M pro , 34.7 kDa) (Supplemental Fig. 1). The concentration of each protein was estimated by bicinchoninic acid protein assay.
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