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. 2022 Aug 11;9:889667. doi: 10.3389/fmolb.2022.889667

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Copyright © 2022 Paliwal, Thom, Hussein, Ravishankar, Wilkes, Charleston and Jones.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The cloning and selection strategies for the generation of the M. bovis recombinant protein atlas. Panel (A). Generic construct design showing the use of either complete or concatenated ORFs tagged at the C-terminus with polyhistidine as present in vector pTriEx1.1. In the event of poor expression the same ORFs were re-cloned with an N-terminal His tag as shown. The sequence of the flexible linker is indicated. Asterisk–stop codon. Panel (B). The protein expression and purification regimen showing the iterative nature of the process. The varied constructs are those with alternate His tags shown in (A). Initial screening was by Western blot for the His tag in all cases.