MBP-FGF2 surfaces were prepared using our previously described method [38 (link)]. Briefly, MBP-FGF2 fusion proteins were obtained from Escherichia coli carrying pMAL-FGF2 plasmids that were generated by inserting human FGF2 cDNA (Bioneer, Korea) into pMAL vectors (New England Biolabs, U.K.). human FGF2 cDNA was cloned from human fibroblasts via polymerase chain reaction (PCR) using oligonucleotide pairs (5′-CCG AAT TCC CCG CCT TGC CCG AGG ATG GC-3′ and 5′-CAA AGC TTT CAG CTC TTA GCA GAC ATT GGA AG-3′; Bioneer) with EcoRI and HindIII restriction sites, respectively. The PCR products were cloned into pGEM-T plasmids (Promega, Madison, WI, USA) to generate pGEM-FGF2. pGEM-FGF2 and pMAL plasmids were digested using EcoRI-HindIII, recovered from an agarose gel, and ligated using a ligation kit (TaKaRa, Shiga, Japan) to generate pMAL-FGF2. MBP-FGF2 (20 μg/mL) spontaneously adsorbed onto polystyrene (PS) surface plates (non-tissue culture-treated 96 or 384 well plates; Falcon, Fisher Scientific, Forest Lawn, NJ, USA) at 37 °C for 4 h.
Pmal vector
Manufactured by New England Biolabs
Sourced in United Kingdom, United States
The PMAL vector is a plasmid-based expression system designed for the production of recombinant proteins in Escherichia coli. It provides a means for cloning and expressing target genes under the control of the strong maltose-inducible PBAD promoter. The PMAL vector includes features such as an N-terminal maltose-binding protein (MBP) tag for enhanced solubility and purification of the expressed protein.
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Lab products found in correlation
Q5 site directed mutagenesis kit, by New England Biolabs (3 mentions)
Amylose resin, by New England Biolabs (2 mentions)
Ligation kit, by Takara Bio (1 mentions)
Human fgf2 cdna, by Bioneer (1 mentions)
Pgem t plasmid, by Promega (1 mentions)
Chicken ovalbumin, by Bio-Rad (1 mentions)
Equine myoglobin, by Bio-Rad (1 mentions)
Ktaexplorer fplc system, by GE Healthcare (1 mentions)
Vitamin b12, by Bio-Rad (1 mentions)
Bovine γ globulin, by Bio-Rad (1 mentions)
Thyroglobulin, by Bio-Rad (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
Mmessage machine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 kit, by Agilent Technologies (1 mentions)
T4 dna polymerase, by New England Biolabs (1 mentions)
Glutathione resin, by GE Healthcare (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Q5 mutagenesis, by New England Biolabs (1 mentions)
Hitrap sp hp, by GE Healthcare (1 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
16 protocols using pmal vector
1
Preparation of MBP-FGF2 Fusion Protein Surfaces
2
Protein Purification and Characterization
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Proteins were purified from soluble fractions in one of two ways. For chimeras containing OspA, the supernatant containing the 6 × -His-tagged protein of interest was purified using an ÄKTA Explorer FPLC system (GE Healthcare) over a Ni2+ Sepharose High-Performance HisTrap HP column (GE Healthcare). For chimeras containing ΔspMBP, purification was performed according to the manufacturer's protocol supplied with pMAL vectors (New England Biolabs). SEC was performed on all 6 × -His-tagged and ΔspMBP-tagged purified proteins. Standards used to calibrate the SEC column were a lyophilized mix of thyroglobulin, bovine γ-globulin, chicken ovalbumin, equine myoglobin and vitamin B12, MW 1,350-670,000, pI 4.5-6.9 (Bio-Rad). Proteins were stored at a final concentration of 1 mg ml−1 in SEC buffer (20 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA pH 8.0) at 4 °C. Expression and purification of EmrE was according to standard protocols43 (link).
3
Enzymatic Preparation of UDP-α-D-Galf
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Chemical reagents including buffer, salts, sugars, NAD+, pyruvate kinase and lactic dehydrogenase enzymes from rabbit muscle were purchased from Sigma-Aldrich. UDP-pyranoses and pNP-sugars were purchased from Carbosynth (Compton, UK), pET-vectors from Novagen and pMal vectors from New England Biolabs. UDP-α-D-Galf was enzymatically prepared, purified and characterized following the procedure previously developed by Prof. Field’s team42 .
4
Functional Characterization of Nagk Kinase
5
ApoE3, ApoE4, and HtrA1 Bacterial Expression
6
Purification of N-terminal His-tagged Mre11 and C-terminal His-tagged Nbs1
7
Recombinant Kinase Protein Assays
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Recombinant proteins were prepared according to (Choi et al., 2017 (link); Lee et al., 2015 (link)), using a modified pMAL vector (New England Biolabs, USA) with maltose binding protein (MBP) and 6X His double tags. Coding regions of relevant proteins were amplified using primers enlisted in Supplementary Table S1 and cloned into the vector. Kinase assays were conducted according to (Choi et al., 2017 (link); Lee et al., 2015 (link)). Site-directed mutagenesis was conducted using the Q5®Site-Directed Mutagenesis Kit (New England Biolabs) according to the supplier’s instruction. Primers are listed in Supplementary Table S1 .
Unless stated otherwise, 0.5 to 1.0 μg of recombinant proteins were used as kinases or substrates. Reactions were conducted at 30°C in a buffer (25 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 μM ATP) containing 2 μCi of [γ-32P]-ATP. After the reaction, the mixtures were separated by SDS-PAGE on 10% to 12% gels, and gels were stained with Coomassie Brilliant Blue R (CBB), dried and autoradiographed.
Unless stated otherwise, 0.5 to 1.0 μg of recombinant proteins were used as kinases or substrates. Reactions were conducted at 30°C in a buffer (25 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 μM ATP) containing 2 μCi of [γ-32P]-ATP. After the reaction, the mixtures were separated by SDS-PAGE on 10% to 12% gels, and gels were stained with Coomassie Brilliant Blue R (CBB), dried and autoradiographed.
8
Cloning and Purification of AnkX and Rab1b
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The AnkX1–800 (referred to as AnkX)–encoding DNA, which previously had been amplified from L. pneumophila genomic DNA (14 (link)), was cloned into a modified pSF vector (Oxford Genetics) by sequence and ligation independent cloning [sequence and ligation independent cloning (SLIC)] using T4 DNA polymerase (New England Biolabs). This resulted in AnkX constructs with an N-terminal decahistidine (His10)–tag, followed by enhanced green fluorescent protein (eGFP) and the tobacco etch virus (TEV) protease cleavage site.
The Rab1b3–174 (referred to as Rab1b)–encoding DNA, which previously had been codon-optimized for expression in Escherichia coli by omitting rare amino acid codons (29 (link)), was cloned into a modified pMAL vector (New England Biolabs) by SLIC using T4 DNA polymerase (New England Biolabs). This resulted in Rab1b constructs with an N-terminal His6-tag, followed by maltose-binding protein (MBP), the TEV protease cleavage site, a Strep-tag, and the PreScission protease cleavage site. For our strategy to purify the AnkXCys:PC:Rab1b complex, the Strep-tag of the Rab1b vector was replaced with a His10-tag by site-specific mutagenesis. All site-specific mutagenesis was performed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).
The Rab1b3–174 (referred to as Rab1b)–encoding DNA, which previously had been codon-optimized for expression in Escherichia coli by omitting rare amino acid codons (29 (link)), was cloned into a modified pMAL vector (New England Biolabs) by SLIC using T4 DNA polymerase (New England Biolabs). This resulted in Rab1b constructs with an N-terminal His6-tag, followed by maltose-binding protein (MBP), the TEV protease cleavage site, a Strep-tag, and the PreScission protease cleavage site. For our strategy to purify the AnkXCys:PC:Rab1b complex, the Strep-tag of the Rab1b vector was replaced with a His10-tag by site-specific mutagenesis. All site-specific mutagenesis was performed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).
9
Purification of Recombinant Spectrin and Protein 4.1R Domains
10
Purification of Recombinant MBP-SYP-1 Protein
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The full-length open reading frame of SYP-1 was amplified from a C. elegans cDNA library and cloned into the pMAL vector (New England BioLabs) to express maltose-binding protein (MBP)–tagged SYP-1 fused to a 6His tag. To make MBP–SYP-1T452A–6His, the Thr at residue 452 was mutated to Ala (ACA → GCA) by Q5 mutagenesis (New England BioLabs). Protein expression was induced at 15°C for ∼16 h with 50 µM IPTG in Rosetta (DE3) pLysS. Bacterial pellets were resuspended in lysis buffer (PBS, 500 mM NaCl, 20 mM imidazole, and 1 mM DTT) and lysed by three freeze/thaw cycles and sonication after lysozyme treatment (0.25 mg/ml) on ice for 30 min. After centrifugation at 15,000 rpm (JA-17) for 30 min, the supernatant was incubated with nickel-nitrilotriacetic acid resins (Qiagen) for 1 h at 4°C. The protein was further purified by HiTrap SP HP (GE Healthcare) using a 100 mM to 1 M NaCl gradient elution in PBS with 1 mM DTT. The peak fraction for MBP–SYP-1 was supplemented with glycerol (20% final) and snap frozen in liquid nitrogen.
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