Production and Purification of Recombinant Antigens

The recombinant antigens were prepared as previously described [11 (link)]. Briefly, the genes that encode antigens EtsC, OmpA, OmpT, and TraT were PCR amplified from χ7122 (etsC, ompT, traT) and RS218 (ompA) strains using primers found in S1 Table, and then cloned into pET-101/D-TOPO^® vectors (Invitrogen). The sequence and the orientation of the genes in the plasmids were verified by sequencing. Recombinant proteins were produced in E. coli BL21, purified via His-tag using ProBond Ni-NTA resin columns (Invitrogen), and then endotoxin was removed using Pierce^™ endotoxin removal spin columns (Thermo Scientific).

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Van Goor A., Stromberg Z.R, & Mellata M. (2017). A recombinant multi-antigen vaccine with broad protection potential against avian pathogenic Escherichia coli. PLoS ONE, 12(8), e0183929.

Publication 2017

pET-101/D-TOPO® vectors ProBond Ni-NTA resin columns

Antigens E coli Endotoxin Genes Plasmids Primers Probond Recombinant proteins Resin Strains Topo Vectors

Corresponding Organization : Iowa State University

Top 5 similar protocols

Variable analysis

independent variables

Genes that encode antigens EtsC, OmpA, OmpT, and TraT

dependent variables

Recombinant antigens

control variables

PCR amplification of genes from χ7122 (etsC, ompT, traT) and RS218 (ompA) strains using specific primers
Cloning of genes into pET-101/D-TOPO® vectors
Verification of sequence and orientation of genes in plasmids by sequencing
Production of recombinant proteins in E. coli BL21
Purification of recombinant proteins via His-tag using ProBond Ni-NTA resin columns
Removal of endotoxin using Pierce™ endotoxin removal spin columns

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