School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemNo Preview AvailableExpression of a recombinant mycobacterial antigen and characterization of fas-defective mutant strains in corynebacterium glutamicum and related speciesChang, Shin-Jae (University of Melbourne, 2003)The research undertaken in this thesis had the initial aim of investigating the utility of Corynebacterium glutamicum species as host for synthesis of foreign proteins of biotechnological importance, given that earlier research had shown that this industrially-important species, normally used for amino acid production, was able to express and secrete a variety of proteins from different bacterial sources. Of particular interest was the use of this species in making proteins that may be secreted or located at the cell surface, where their expression may have been important in antigenicity or other physiological functions: proteins made and secreted by related organisms, including pathogenic Mycobacterium species, were of particular interest because many pathogens cannot he cultured readily in the laboratory environment or exist as obligate intracellular parasites. It was also likely that the C. glutamicum and Mycobacterium species would share functionality in their cell surface proteins, an assumption based on their common synthesis of mycolic acids complex surface lipids that are involved in making cells from this group resistant to chemical and physical assault. The first part of the thesis describes a study on the synthesis and secretion of the 85 antigen complex, component of which are important immunogens in Mycobacterium and are secreted proteins with activity involved in the attachment of mycolic acids to the cell wall. During the course of this study, PCR amplification using primer sets based on the 85B antigen complex from M. bovis was followed up by sequencing the PCR products. Rather than corresponding to an 85B homologue, the DNA had high sequence similarity to a fatty acid synthase (FAS) gene previously found in Brevibacterium (now Corynebacterium) ammoniagenes. The subsequent focus of the thesis was on characterization of the presumptive fas gene in C. glutamicum in terms of gene inactivation and impact on lipid synthesis, for both fatty acids and mycolic acids. The first part of this project was to undertake controls for later experiments if the mycobacterial antigen 85 complex components (A, B and C) or any homologue of these were present in Corynebacterium glutamicum AS019 and its related strains, C. glutamicum ATCC13032, C. glutamicum CG2, C. lactofermentum BL1 and C. flavum BF4. This involved determining on the expression of a recombinant antigen 85 complex in C. glutamicum AS019 and related cell-surface mutants (MLB 133 and MLB 194). In order to detect the presence of the mycobacterial antigen 85 complex (A, B and C) or homologues, several experiments, including Polymerase Chain Reaction (PCR), Southern hybridization and Western blotting experiments, were carried out. The screening focused on the antigen 85B as this component is the most abundant of the antigen 85 complex in Mycobacterium species and there is considerable similarity between antigen 85 complex members at both nucleotide and amino acid sequence levels. Furthermore, antigen 85B was most extensively studied in the literature so that the materials needed to study this (such as PCR primers, antibody and probes for Southern hybridization experiments) were easier to make or obtain from other research groups. A PCR Mal 1/21 primer set was designed based on the most conservative regions of the antigen 85B from several species of Mycobacterium. Amplification produced several feint products from C. glutamicum AS019, C. lactofermentum BL1 and C. flavum BF4, which was an unexpected outcome. All the major PCR products were cloned into the pGEM�T vector and sequenced to determine similarity with the sequence of mycobacterial antigen 85 complex. However, the �0.8 kb PCR products from C. glutamicum AS019 and C. lactofermentum BL1 showed relatively high similarity (-70%) with a fatty acid synthase gene (fasA) of C. ammoniagenes and �60% with the fasA found in M. tuberculosis. Similar PCR products were found in all of the C. glutamicum strains tested. In Southern hybridization experiments, two different probes were used for more intensive analysis. One was the 0.8 kb PCR product produced by the Mal 1/21 primer set when pGEX-MPB59 (an expression vector which contains a mycobacterial antigen 85B gene) was used as template. The other was a 0.49 kb fragment produced by PstI/Xhol double-digestion of the above 0.8 kb PCR product. Relatively lower prehybridization/hybridization temperature (55�C) and longer expose time were employed to detect any loosely-bound signal, however, no specific signal was detected using gDNA from C. glutamicum AS019 and its related strains. In Western blotting experiments, anti-antigen 85B antibody raised in rabbits was used as the primary antibody to detect any signal from whole disrupted cells and culture filtrate of C. glutamicum AS019 and its related strains. No signal was found using culture filtrates concentrated 50 fold, however, a signal was found using disrupted cells of all strains. To determine the location of this protein, cell wall proteins were extracted from whole cells with 50 mM Tris buffer containing 2% Sodium Dodecyl Sulfate (SDS). No signal was found from cell wall extracts so that this protein may be located in the inner membrane or cytosol. The size of this protein was almost 40 kDa which is significantly bigger than the mycobacterial antigen 85 complex (B is 32 kDa and A and C are 30 kDa). Even though a signal was found from Western blotting experiments, it was concluded that there was no antigen 85 complex homologues present in C. glutamicum due to the negative results from PCR and Southern hybridization experiments. Several strains were then transformed with pCGL1055, which contains the antigen 85A gene, to determine the secretion level of recombinant antigen 85A in C. glutamicum AS019, C. glutamicum MLB133, C. glutamicum MLB194, C. lactofermentum BL1 and C. flavum BF4. Since pCGL1055 had the cspB promotor, two sets of primer were used to confirm the presence of the cspB gene (coding for PS2 protein in corynebacteria) in the strains used in this expression work. To quantify the amount of secreted recombinant antigen 85A, recombinant antigen 85B was prepared as the standard using Glutathione Sepharose 4B� affinity chromatography followed by thrombin cleavage and gel filtration chromatography using material prepared from E. coli TG2 transformed with pGEX-MPB59, the expression vector which contains the antigen 85B gene. C. glutamicum MLB 133 and MLB 194 showed relatively higher secretion level than the parent strain, C. glutamicum AS019, which suggests that the changes in the cell wall structure of the mutants caused better secretion of the foreign recombinant protein. C. flavum BF4 also showed relatively higher secretion levels than AS019. Volumetric and cellular productivity for the expressed recombinant antigen 85A from several corynebacterial strains were calculated to determine the efficiency of strains in secreting the protein into culture fluids. The amount of 85A protein secreted was expressed as the equivalent of 85B protein measured in parallel using anti-antigen 85B. In volumetric productivity from C. glutamicum strains AS019 (spontaneous Rif mutant of C. glutamicum ATCC 13059), MLB 133 and MLB 194 (the cell wall modified mutants of C. glutamicum ATCC 13059) was 16-17 ng/ml. However, C. lactofermentum BL1 and C. flavum BF4 expressed relatively higher amounts of antigen 85A (22-23 ng/ml) than the above strains. Cellular productivity was determined by calculating the amount of antigen 85A secreted from the given number of cells of each transformant by adjusting the optical density of each overnight cultured culture filtrate to 100 at 600nm. The cell wall modified mutants, C. glutamicum MLB 133 and C. glutamicum MLB194, showed higher efficacy (730-775 ng/ml) in expressing antigen 85A from the given number of cells than its wild-type strain, C. glutamicum AS019 (440 ng/ml). C. flavum BF4 showed almost the same efficiency (about 750 ng/ml) as the mutants. The new finding of a fas-gene homologue using PCR amplification prompted further studies aimed at characterizing its role in synthesis of fatty acids and mycolic acids in C. glutamicum. The inactivation of a gene by homologous recombination was used to characterize the fas-gene homologue, employing the 0.8 kb fragment found in the previous PCR experiments. The 0.65 kb fragment, the nested PCR product from the above 0.8 kb fragment, was amplified using the fas11/21-EcoR I-1 primer set. The amplified 0.65 kb fragment was digested with EcoR I and cloned into pECMA, a suicide vector that does not replicate in Corynebacterium strains without homologous recombination between the insert and recipient chromosomal DNA due to the lack of a replication origin on the vector. The newly constructed suicide vector, pECMAfas-AS019, was introduced into the donor strain, E. coli S17-1, a mobilizing strain constructed by the integration of self-transmissible (Tra') plasmid, RP4 2-Tc