Bovine tuberculosis caused by Mycobacterium bovis, is a significant global pathogen causing economic loss in livestock and zoonotic TB in man. Several vaccine approaches are in development including reverse vaccinology which uses an unbiased approach to select open reading frames (ORF) of potential vaccine candidates, produce them as recombinant proteins and assesses their immunogenicity by direct immunization. To provide feasibility data for this approach we have cloned and expressed 123 ORFs from the M. bovis genome, using a mixture of E. coli and insect cell expression. We used a concatenated open reading frames design to reduce the number of clones required and single chain fusion proteins for protein pairs known to interact, such as the members of the PPE-PE family. Over 60% of clones showed soluble expression in one or the other host and most allowed rapid purification of the tagged bTB protein from the host cell background. The catalogue of recombinant proteins represents a resource that may be suitable for test immunisations in the development of an effective bTB vaccine.