Establishment Of Effective Microbial Expression System For Plectasin And T4 Lysozyme

This paper attempted to express the low molecular weight antimicrobial peptide～plectasin in Pichia pastoris. Firstly, the gene encoding mature plectasin was synthesized according to yeast codon usage bias, and then directionally inserted into the MCS of the expression vector pPIC9K with restriction endonuclease and T4 ligase, which is inducible expression with methanol as the inducer in P. Pastoris. In order to express efficiently the gene encoding heterologuos low molecular weight of protein in P. Pastoris, an artificial enhancer with 3 times tandem repeating sequence, derived from TYMV3'UTR and CMVp enhancers was inserted into the downstream of terminator codons of the plectasin gene, forming a new inductive and secretary expression vector of P. Pastoris designated 3ENple-9k. The 3ENple-9K plasmid DNA linearized with Sal I was transformed into the GS115 strain of P.pastoris. By using selective medium, G418 resistance and shaking-flask culture, one transformant designated PleI was selected from hundreds of transformants, which can express effectively and stably the recombinant plectasin peptides. Based on the pilot test (50L fermenter), a lot of parameter relating to the conditions of fermentation and protein expression were optimized, including nutrient component, temperature, cell density, pH, dissolved oxygen, inoculum size, methanol concentration and inductive time. The expression level up to 0.14g/L of the recombinant plectasin was realized by using the strategy motioned above. In addition, an extracting and purifying process of the recombinant plectasin was investigated and a simple, convenient and economic method was established, which involved in four stages: 1) pretreatment of the fermentation broth; 2) retreatment of the supernatant of fermentation; 3) high enrichment of the supernatant of fermentation by nano-filtration; 4) high purification of the recombinant protein by molecular-sieve chromatography. Meanwhile, conditions of experimental material and methods affecting recovery of the recombinant plectasin were studied. Antibacterial activity analysis of the recombinant plectasin in vitro shows that not only can it inhibit strongly growth of some common gram-positive pathogenic bacteria, but also inhibit some gram-negtive bacteria. In vivo inhibiting experiment indicated that the recombinant plectasin are able to partly cure the infection of mice caused by Streptococcus Equi Subsp. Zooepidemicus.A modified recombinant T4 lysozyme was firstly attempted to express and produce in Hansenula polymorpha. The gene encoding the modified T4 lysozyme according to yeast codon usage bias was inserted directionally into the MCS of a modified expression vector pPIC9K containing the rDNA sequence from H.polymorpha as a homologous recombination sequence, which is inducible expression with methanol as the inducer in H. polymorpha. The expression vector designated T4-rD-9K was linearized and transformed into the A16 strain of H.polymorpha. By using selective medium G418 resistance and shaking-flask culture, one transformant designated H5 was selected from hundreds of transformants, which can express effectively and stably the recombinant T4 lysozyme. Based on the results mentioned above, a dual-gene expression system designated pleT4-9K was constructed and transformed into the GS115 strain of P.pastoris. One transformant named PTI was obtained by screening, which can simultaneously express both the plectasin and T4 lysozyme genes in P.pastoris. Antibacterial activity analysis in vitro of the fermentation broth shows that both the recombinant proteins can play a role simultaneously, which predicts a broad application of the recombinant proteins at medicine, food, forage industry field in the near future.For further increase expression efficiency of the heterologous genes in host strain,a high expression system was also constructed, which will be used in Trichoderma reesie. The Pcbh1-sig and Tcbh1 DNA sequences were prepared by PCR approach, using genomic DNA of T. ressei as the template. The Pcbh1-sig and Tcbh1 sequences were added to 5'end and 3'end of the gene encoding plectasin or T4 lysozyme respectively, which formed two new expression cassettes. The plectasin or T4 lysozyme expression cassette was into the MCS of a modified expression vector pPIC9K containing a hygromycin resistance gene as the screening marker, forming two expression vectors designated T4-hT-9K and ple-hT-9K, which are inductive and secretary with cellulose as the inducer in T. reesie.