Expression of T4 lysozyme gene in escherichia coli and trichoderma reesei

Abstract

The T4 lysozyme, from bacteriophage T4, has the ability to lyse bacterial cell walls; thus it has attracted immense attention as an antimicrobial agent. Nonetheless, the production of T4L cannot meet our needs. All of the work presented here in this thesis, focuses on the potential exploitation of synthetic gene-coding T4L protein to improve the economic feasibility of antimicrobial agent production by producing a natural antimicrobial agent. Synthetic t4l was constructed to enhance the production of recombinant T4L for industrialization purposes after its successful expression in the two hosts. Therefore, comparing the wild type and synthetic t4l from nucleotide sequences until protein 3D structure was unavoidable. Two T4L genes that have been studied are the wild type and the synthetic one. The later, new synthetic (t4l) gene was produced by using codon-based modification with alternation of triplet nucleotides that encodes two specific amino acid residues at position 6 and 12 in a polypeptide chain of T4L protein. Wild type and synthetic t4l have variants in the size of nucleotides, 459 bp and 491 bp, respectively, but both have equal amino acids size, 164 a.a. The comparison of 3D protein structure showed us how both protein structures have structural similarity by calculating RSMD; 0.17 for backbone coordinate and 0.14 for carbon alpha coordinate. Also, structure sequence identity was 98.87% by using the alignment of editor Jalview. Additionally, developed T4L gene with codon-based modification has been cloned into three vectors with different promoters in order to be expressed in selected strain of E. coli then T. reesei. Expression of a synthetic t4l by using E. coli system was by transforming of the three plasmids-bearing-synthetic t4l separately, into two strains of E. coli, JM109 and BL21(DE3)pLysS. The later had been tested for its sensitivity and/or resistance to the lysis action of lysozyme enzyme before the transformation-taking place by using agar halo assay. Resistance of BL21(DE3)pLysS strain to lysozyme activity has been confirmed with our halo assays, which keep this strain to be a good choice for expression of this lysis enzyme. Also, the results of both enzyme digestion and PCR showed that the synthetic t4l had successfully transformed into selected E. coli strain. Furthermore, the expression of that gene had been showed on SDS-PAGE assay with an apparent molecular mass of approximately 18 kDa. However, determine and measure the recombinant T4L production is still needs more research to work on it. Additionally, T. reesei has the capacity to produce extracellular enzymes in very large amounts. Invention relates method for efficiently expressing and producing recombinant protein of T4L from T. reesei QM6a by using protoplast-based transformation method. T4 lysozyme synthesis operon (t4l) cloned in three different plasmids was successfully integrated in T. reesei QM6a genome, to form recombinant strains, termed as ST4L1, ST4L2, and ST4L3. T4 lysozyme encoded by the t4l was successfully expressed in two of our recombinant strains, which are ST4L2 and ST4L3, as confirmed by SDS-PAGE. We successfully constructed a recombinant T. reesei strain expressing exogenous gene, specifically t4l that originated from phage T4 and integrated into the genome. Recombinant T4L was stably produced by those strains with a selected antibiotic supplementation. We develop a novel system by which foreign gene can be successfully inserted into the chromosome of T. reesei, where transcription can be driven by cbhI promoter, with the need for addition of inducer that is carbon source. T4 lysozyme production by this system was stable and the system may be useful in biotechnological fermentation processes. Success of such a technology will facilitate production of T4L for industrialization and medical purposes.