The National Center for Biotechnology Information file for C. neoformans var. grubii H99 strain (taxid:235443) served as the source for cDNA and protein sequences of Cda1 (CNAG_05799), Cda2 (CNAG_01230), Cda3 (CNAG_01239), and Fpd1 (CNAG_06291). cDNAs for these proteins and the mutated versions of Cda2 (Cda2-M1 and Cda2-M2) were synthesized and cloned into pET19b (GenScript) so that the vector-encoded His tag was integrated with the N terminus of the cDNA. Recombinant protein was made in E. coli strain BL21(DE3) (New England BioLabs) using Overnite Express TB medium (Novagen) and purified on His·Bind resin (EMD Millipore) in the presence of 6 M urea, as described previously (16 (link)). Following elution with imidazole, proteins were dialyzed against 6 M urea/20 mM Tris-HCl, pH 7.9, and concentrated to 10 mg/mL using Amicon Ultra-15 centrifugal filters (10-kDa cutoff; Merck Millipore). The protein concentration was determined by the bicinchoninic acid (BCA) assay. To assess purity, the recombinant proteins were resolved by SDS-PAGE and stained with Coomassie InstantBlue (Expedeon, Ltd.).
Pet19b
Manufactured by GenScript
Sourced in United States
PET19b is a plasmid vector designed for the expression and purification of recombinant proteins in Escherichia coli. It contains a T7 promoter for high-level protein expression and a his-tag for simple affinity purification of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
His bind resin, by Merck Group (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (2 mentions)
Bl21 de3, by New England Biolabs (1 mentions)
Pet30a, by GenScript (1 mentions)
Coomassie instant blue, by Abcam (1 mentions)
Shuffle t7 competent e coli cells, by New England Biolabs (1 mentions)
5 protocols using pet19b
1
Recombinant Chitin Deacetylase Proteins
2
Recombinant Lyme Disease Enzymes
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The optimized B. burgdorferi genes of L -lactate dehydrogenase (LDH/bb0087), GAP dehydrogenase (gapdh/bb0057) and Enolase (eno/bb0337) were cloned into pET30a (LDH and GAPDH) or pET19b (Eno) by GenScript, Piscataway, NJ, United States. LDH and GAPDH were overexpressed and purified by GenScript. Eno was overexpressed and purified as previously described. Briefly, E. coli BL21 (DE3) was transformed with the appropriate vector. Transformants were grown in LB at 37°C for LDH and GAPDH and at 18°C for Eno and induced by adding 1 mmol IPTG. Purification was performed using a nickel affinity column and protein concentrations were determined using a PierceTM BCA Protein Assay Kit (23225, Thermo Scientific, Rockford, IL, United States) with BSA as a standard.
3
Recombinant Protein Purification Protocol
4
Heterologous Expression of Rbe1p and Rbt4p in E. coli
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For heterologous expression of Rbe1p and Rbt4p in E. coli, RBE1 and RBT4 gene sequences were codon optimized (S7 Fig ), chemically synthesized with additional XhoI and BamHI restriction sites and cloned into vector pET-19b(+) (GenScript, Piscataway, USA). Transformation of SHuffle® T7 Competent E. coli cells (New England Biolabs) with the plasmids was done using the method described by Chung et al. [15 (link)].
A list of all primer sequences and constructed plasmids is found in the supplementary information (S2 and S3 Tables).
A list of all primer sequences and constructed plasmids is found in the supplementary information (
5
Recombinant Protein Purification from Yeast
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The glucan particles were prepared from Fleischmann’s Baker’s yeast (AB Mauri Food Inc., Chesterfield, MO, USA), as previously described [8 (link)]. The Cda2 gene from C. neoformans was synthesized and cloned into pET19b (GenScript Biotech, Piscataway, NJ, USA) so that the vector-encoded His tag was integrated with the N terminus of the cDNA. The recombinant protein was made in the E. coli strain BL21(DE3) (New England Biolabs, Ipswich, MA, USA) and purified on His·Bind resin (EMD Millipore, Burlington, MA, USA), as previously described [24 (link)]. All of the reagents, solvents, and buffer solutions were purchased from Sigma-Aldrich (Burlington, MA, USA), unless specified otherwise, and used without further purification. The tissue culture materials were purchased from Gibco (Waltham, MA, USA).
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