Recombinant His-tagged merozoite surface protein-1 (MSP-142) (amino acids 1398–1754; Accession P13828) and apical merozoite antigen-1 (AMA-1) (amino acids 26–478; Accession AAC47193) proteins from P. yoelii 17X were produced in E. coli as previously described.45 (link),46 (link) After purification and refolding the MSP-142 and AMA-1 proteins, the His-tag was cleaved using a Thrombin kit (Millipore Sigma, St. Louis, MO). Confirmation of the His-tag removal was confirmed by W. blot using an anti-His Ab (ThermoFisher). MSP-142 and AMA-1 were biotinylated and tetramerized with streptavidin-PE (SA-PE; Agilent, Santa Clara, CA) as previously described.47 A decoy reagent to gate out the non-MSP-142 or AMA-1–specific B cells was constructed by conjugating SA-PE to DyLight 650 (ThermoFisher) followed by washing and removal of any unbound DyLight 650 and incubating with an excess of biotin as described.47
Anti his ab
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
The Anti-His Ab is a laboratory reagent used for the detection and purification of recombinant proteins that contain a histidine tag. It provides a simple and effective method for identifying and isolating these tagged proteins from complex mixtures.
Automatically generated - may contain errors
4 protocols using anti his ab
1
Recombinant Malaria Antigen Production and Characterization
2
Recombinant protein production and biotinylation
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Recombinant His-tagged merozoite surface protein-1 (MSP-142) (amino acids 1398-1754; Accession P13828) and apical merozoite antigen-1 (AMA-1) (amino acids 26-478; Accession AAC47193) proteins from P. yoelii 17X were produced in E. coli as previously described. 44, 45 After purification and refolding the MSP-142 and AMA-1 proteins, the His-tag was cleaved using a Thrombin kit (Millipore Sigma, St. Louis, MO). Confirmation of the His-tag removal was confirmed by W. blot using an anti-His Ab (ThermoFisher). MSP-142 and AMA-1 were biotinylated and tetramerized with streptavidin-PE (SA-PE; Agilent, Santa Clara, CA) as previously described. 46 A decoy reagent to gate out the non-MSP-142 or AMA-1-specific B cells was constructed by conjugating SA-PE to DyLight 650 (ThermoFisher) followed by washing and removal of any unbound DyLight 650 and incubating with an excess of biotin as described. 46
3
Profiling Mycobacterium tuberculosis Antigens
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A total of 59 Mtb recombinant proteins, previously identified by different Mtb antigen discovery approaches (13 (link)), were tested in this study (Table 2 ). As described previously (11 (link)), Mtb genes were amplified by PCR from genomic H37Rv DNA and cloned by Gateway technology (Invitrogen, Carlsbad, CA, USA) in a bacterial expression vector containing, overexpressed in Escherichia coli (E. coli) BL21 (DE3) and purified. Gel electrophoresis and western blotting with an anti-His Ab (Invitrogen) and an anti-E. coli polyclonal antibody (a gift of Statens Serum Institute, Copenhagen, Denmark) were used to check the size and purity of the recombinant proteins. Rv0287-Rv0288, Rv2346c-Rv2347, and Rv3614-Rv3615 were produced as fusion proteins to mirror the pairwise dependent secretion pathway followed by T7S systems. All recombinant proteins were tested to exclude protein-non-specific T cell stimulation and cellular toxicity (11 (link)).
4
Recombinant Protein Production and Purification
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As described previously86 (link), Mtb genes were amplified by PCR from genomic H37Rv DNA and cloned by Gateway technology (Invitrogen, Carlsbad, CA, USA) in a bacterial expression vector containing a histidine (His) tag at the N-terminus. Vectors were overexpressed in Escherichia coli (E. coli) BL21 (DE3) and purified. The size and purity of the recombinant proteins were analysed by gel electrophoresis and western blotting with an anti-His Ab (Invitrogen) and an anti-E. coli polyclonal Ab (gift of Statens Serum Institute, Copenhagen, Denmark). Rv0287-Rv0288, Rv2346c-Rv2347, and Rv3614-Rv3615 were prepared as fusion proteins29 (link) to mimic the pairwise dependent secretion pathway followed by T7S systems87 (link). At this point, two proteins (Rv1197 and Rv1831) were excluded due to problems linked to their production. Forty-eight recombinant proteins were produced and tested to exclude protein-nonspecific T-cell stimulation and cellular toxicity50 (link).
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