Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

Abstract

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.

Keywords: Mycobacterium leprae; Mycobacterium smegmatis; recombinant protein; Mycobacterium tuberculosis; expression system; protein production; shuttle vectors.

Figure 1

Protein structures present in the Protein Data Bank from different mycobacterial species. The numbers indicate discrete entries with <90% sequence identity. For clarity, the figure does not include species for which only two (M. xenopi and M. fortuitum) or one (M. gastri, M. goodie, M. vanbaalenii, M. sp JLS, and M. sp JC1) entry is found.

Figure 2

Crystal structure of Mtb proteins expressed using alternative expression hosts. All proteins were expressed in M. smegmatis expression host, except for 1GN2 (M. vaccae), 4JN6 (Rhodococcus jostii), and 2VF2 (Pseudomonas putida).