Prokaryotic Expression, Purification Of Thanatin And Testing Of Its Antibacterial Activity

Background:Thanatin is an inducible insect peptide firstly isolated from the hemolymph of Podisus maculiventris, related to the other antimicrobial peptides such as brevinins, protegrins, and tachyplesins. It was reported as an antibacterial peptide with a broad spectrum against G+(Gram-positive), G- (Gram-negative) bacteria and fungi under physiological concentrations, and is active against multidrug resistant clinical isolates of Enterobacter aerogenes and Klebsiella pneumoniae.The Thanatin transgened rice showed sufficient level of resistance to the rice blast fungus Magnaporthe oryzae, presumably due to the repressive activity of Thanatin to its initial stage of infection.Thanatin is composed of 21 Amino Acids (GSKKPVPIIYCNRRTGKCQRM). Its center is a P-sheet, which is depend on the disulfide bond formed between C11 and C18.The C-terminal of this peptide is highly positive charged and has a disulfide loop, which is very similar to the frog skin secretion antimicrobial peptides of the brevinin family.Structure-activity research revealed that the bioactivity of Thanatin was highly dependent on four structures:1.C terminal loop;2.the last 3 amino acids in C-terminal;3.the 7 hydrophobic amino acids in N-terminal;4.the first 3 amino acids in N-terminal. The last 3 amino acids are mostly responsible for the anti-bacterial activity to G+ bacteria, and some G" bacteria. The first 3 amino acids in N-terminal are crucial to the reverse paralleled P-sheet stability, and it is essential for anti-fungi activity.A ramdomly mutated Thanatin sequences were used to analyzed the structure-activity relation. The result suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining theβ-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.The chemically modified Thanatins with the methyl group (CH3), ethyl group (C2H5), and normal-octyl group (C8H17) at the side-chain of cysteine residues were synthesized, and used for anti-bacterial activity comparison. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteus than wild type Thanatin, which suggested an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues.The tert-butyl group at two cysteine residues which form an intramoleular disulfide bridge in Thanatin peptide was modified to be C11tBu/C18tBu, exhibited higher antimicrobial activity than natural original one toward G+ bacteria Micrococcus luteus, whereas lower activity toward G" Escherichia coli. This finding suggests that disulfide-bridge formation is not only indispensable for exhibition of antimicrobial activity of Thanatin but also closely related to the activity specificity towards bacteria. Thanatin acts against E. coli stereospecifically by taking advantage of its C-terminalβ-hairpin structure, while the activity against M. luteus does not relate to structures and correlates very well to side-chain hydrophobicity.The limited source of native antibacterial peptides, and the difficulty in extraction and purification restrict the using of antibacterial peptides in medicine, food industry, animal husbandry and agriculture.It is essential to produce antibacterial peptide with genetic methods, such as expressing it in E. coli, taking the advantage of high efficiency and low cost. Even though, the expression of antibacterial peptides in E. coli is facing a lot of challenges, such as the toxity of antibacterial peptides to host cells, the tendency to be degraded by proteases and so on. That is why antibacterial peptides are usually expressed as a fusion protein, to improve its stability in host cells.Nevertheless, there bring a lot of difficulties to get rid of the fusion protein from the antibacterial peptides. The recombinant fusion protein is usually digested with bromine cyanide, Hydroxylamine and Enterokinase to get rid of the fusion tag from the target protein. This method will leave some extra amino acid residues in the terminal, affecting the native composition and structure of antibacterial peptides, and causing the reducing or lost of antibacterial activity. Furthermore, antibacterial amino acids composition is between 12 to 50, and the molecular weight is about 4kDa, so it is very difficult to get rid of those proteases from the small peptides.As Thanatin is a simple peptide without any complicated structure, E. coli expressing Thanatin will keep its native amino acids sequence and protein folding structure.Our research is to analyze the antibacterail activity of the Thanatin expressed by E. coli ER2566,which originally fusion-expressed with intein, a kind of self-cleavage protein to cleave between intein and Thanatin to separate them efficiently. In this research, we put Thanatin gene just after C-terminal of intein, constructed pTYB11-Thanatin plasmid.Then the plasmid was transformed into E. coli ER2566, and induced with IPTG to express. As intein can bind specificly with Chitin, after affinity chromatography,the intein-Thanatin was bound on the Chitin column. DTT and cysteine was used for inducing intein self-cleavage, the target protein was separated with intein and released from the column. The target protein was eluted with the elution buffer. The eluted Thanatin has the same amino acids sequence as native Thanatin without adding any extra amino acid to both N-and C-terminals, so that the antibacterial activity can be retain as best as possible. The whole purification process do not need any proteases.Escherichia coli is a kind of normal indigenous flora which abundantly exists in the intestine. It has become an important infectious pathogen in hospitals, with the abuse use of antibiotics such as the 3rd-generation cephalosporin. Escherichia coli has produced serious drug resistance to antibiotics due to the existance of ESBLs(Extended-Spectrumβ-Lactamases),resulted in increased morbidity, mortality and cost in treating the infections they cause.ESBLs is a kind of plasmid-introduced hydrolase, which can hydrolyse the 1st and 2nd-generation cephalosporins, penicillin, broad-spectrum cephalosporin, and monoamide antibiotics. ESBLs activity can be inhibited by inhibitors like clavulanate and salbactam. The existance of ESBLs is the most important mechanism of drug resistance toβ-Lactamases antibiotics for G- bacteria. Mostly, the ESBL bearing plasmids also contain the other drug-resistance genes such as aminoglycoside antibiotics resistance gene, so it is easy to produce multidrug resistance for the G-negative bacteria, especialy for Escherichia coli and Klebsiella pneumoniae. Under the antibiotics select pressure, the ESBL bacteria will generate new drug-resistance mechanism and spread rapidly between different strains by plasmid transformation, comissure and transduction, which will result in serious outbreak of infection in hospitals. It is meaningful to produce some new antibiotics or substitutes with different antibacterial mechanism. Thanatin, as an antibacterial peptide with the antibacterial mechanism of breath inhibition, and shew its advantages in killing ESBLs bacteria, could be a potential antibiotics substitute in the future.Objectives:1.Expressing Thanatin in prokaryotic expression system and purification of the recombinant protein;2.Antibacterial activity assay for the recombinant Thanatin expressed by E.coli; 3.Evaluation of the application potential for the purified recombinant Thanatin expressed by E.coli.Methods:1.The Thanatin gene was modified using the Escherichia coli preferred codons synthesized by Overlapping PCR.2.The Thanatin gene was cloned into pMD 18-T vector to construct the recombinant plasmid pMD 18-T-Thanatin and identified by sequencing.3.Thanatin was fusion with intein and cloned into pTYB11 plasmid, The recombinant plasmid pTYB11-Thanatin was identified by sequencing.4.pTYB 11-Thanatin plasmid was transformed into E.coli ER2566 and inducing by IPTG.5.Optimized the condition of expression the Thanatin-Intein fusion protein.6.Purify Thanatin by inducing self-cleavage activity and seperate Thanatin protein from intein.7.The antibacterial activity of Thanatin was determined by agarose diffusion assay with Pseudomonas aeruginosa, Drug-resistance Enterotoxigenic Escherichia, Escherichia coli 0157, Klebsiella pneumoniae,Shigella flexneri, Shigella snnei,Candida albicans and ESBLs E. coli.8.To compare the difference between the sensitive and drug resistant E. coli as for the Thanatin antibacterial activity measured by the inhibition zone diameter, Statistical software SPSS 13.0 was applied and analysis of two-sample T test were adopted.Results:1.Thanatin gene was cloned and recombinant expression plasmid pTYB11-Thanatin was successfully constructed.2.After IPTG inducing, Thanatin-Intein fusion protein was expressed as a soluble protein in E. coli ER2566 cytosol.3.The Thanatin was purified by Chitin column, the cleaved Thanatin was eluted with a protein concentration of 245ug/ml at the first 4ml.4.The antibacterial activity assay results demonstrated that purified Thanatin was apparently active against clinical G- bateria and expecially showed advantage in killing drug-resistant bacteria, such as Drug-resistant Shigella flexneri, Shigella snnei, Xanthomonas maltophilia pneumonia, Enterotoxigenic Escherichia, Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli 0157, and ESBLs producing Escherichia coli.5.80 strains of drug resistant (ESBL producing) and 30 strains of sensitive E. coli, were used for anti-bacterial assay.There was no significant difference between the sensitive and drug resistant E. coli as for the Thanatin antibacterial activity measured by the inhibition zone diameter(cm) (P>0.05,t-test).Conclusion:1.Thanatin gene was subcloned into pTYB11 downstream of intein, a kind of protein splicing element which have inducible self-cleavage activity.The recombinant plasmid was transformed into E.coli ER2566.After induction with IPTG, the recombinant Thanatin was highly expressed as a soluble protein.2.The purified Thanatin had a very good antibacterial activity against G-bacteria, especially shew its advantages in overcoming drug-resistance, actively killing drug-resistant ESBLs bacteria.3.Thanatin has the potential to be developed into an antibiotics substitute in the future.