title: Cloning, expression and rapid purification of active recombinant mycothiol ligase as B1 immunoglobulin binding domain of streptococcal protein G, glutathione-S-transferase and maltose binding protein fusion proteins in Mycobacterium smegmatis.
abstract: Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol found in Mycobacteria spp. and other actinomycetes. Mycothiol plays a fundamental role in these organisms by helping to provide protection from the effects of reactive oxygen species and electrophiles, including many antibiotics. It has recently been demonstrated that the MshC gene and more generally the production of mycothiol are essential to Mycobacterium tuberculosis, indicating that MshC may represent a novel target for new classes of antituberculars. Because MshC cannot be expressed heterologously in Escherichia coli and isolation from Mycobacterium smegmatis is impractical, we have optimized the E. coli-M. smegmatis shuttle vector pACE for cloning and recombinant expression of MshC (under control of an acetamidase-inducible promoter). To improve expression levels and simplify purification, we further constructed three N-terminal-MshC fusion proteins where N-terminal tags included the B1 domain of streptococcal protein G (to give GB1-MshC), glutathione-S-transferase (to give GST-MshC) and maltose binding protein (to give MBP-MshC), for expression in M. smegmatis. By expressing all three fusion proteins in a mutant strain of M. smegmatis mc(2)155, namely I64 L205P MshC M. smegmatis which lacks mycothiol ligase activity, we demonstrate in vivo mycothiol ligase activity for each construct. Recombinant GST-MshC and MBP-MshC were isolated in one step by affinity chromatography in a yield of 0.7 and 1.2 mg fusion protein/L and exhibited specific activities of 9 nmolmin(-1)mg(-1) and 25 nmolmin(-1)mg(-1), respectively.
authors.affiliation: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bldg 8, Room 1A-02, Bethesda, MD 20892-0820, USA.
authors.forename: Maria-Teresa
authors.initials: MT
authors.lastname: Gutierrez-Lugo
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Newton
authors.forename: Robert C
authors.initials: RC
authors.lastname: Fahey
authors.forename: Carole A
authors.initials: CA
authors.lastname: Bewley
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002352:Carrier Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D003545:Cysteine / Q000096:biosynthesis* / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry*; D006020:Glycopeptides / Q000096:biosynthesis* / Q000737:chemistry; D007136:Immunoglobulins / Q000737:chemistry; D007294:Inositol / Q000096:biosynthesis* / Q000737:chemistry; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D058619:Maltose-Binding Proteins; D020102:Mycobacterium smegmatis / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D006020:Glycopeptides; C034284:IgG Fc-binding protein, Streptococcus; D007136:Immunoglobulins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; C089265:mycothiol; D007294:Inositol; D005982:Glutathione Transferase; D008025:Ligases; D003545:Cysteine