| [Backgroud]With the widespread application of broad-spectrum antibiotics in clinic, more and more bacterium develops drug resistance, which hinders the treatment of diseases and increases the social expenses. Prolonged use and abuse of antibiotics serve as bacterial stress responses and determinants of antimicrobial resistance, and cause many serious adverse effects, such as liver and kidney function damage, hematologic toxcity and neurotoxicity, gastrointestinal adverse reaction and so on. The adverse effects also limit the application of sensitive drugs to the treatment of infectious diseases caused by drug-resistant Gram-positive bacterium. Therefore, developing novel antimicrobial agents which are effictive are urgently needed.Antibacterial peptides (AMPs), an important component of the innate immune of animals, has been proved to be a kind of peptides with features of higher antibacterial activity, higher antiviral activity and immune regulatory function. Researches showed that AMPs were widely disturbed in various organisms and had high-efficiency activity against bacteria, fungi, viruses, protozoa, even cancer cells. Compared with conventional antibiotics, AMPs provides unique mechanism and avoides drug resistance, and even some kinds of AMPs have immune regulatory function. Today many kinds of AMPs have been used in clinical trials. Pexiganan, one kind of AMPs which was developed by Magainin company, has showed efficacy in the treatment of infectious diabetic feet. Plectasin was the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella in 2002. The AMPs was especially active against a broad spectrum of Streptococcus Pneumoniae, Staphylococcus aureus, Enterococci, including Penicillin-Resistant Streptococcus Pneumoniae (PRSP) and Methicillin-resistant Staphylococcus aureus (MRSA), but brought little toxcity. Given these characteristics, Plectasin becomes an attractive candidate to serve as a kind of novel antibiotic and shows a broad application prospect.The content of natural AMPs in creatures is very low. However, isolating from organisms or chemical synthesis is a complex and costly exercise with no guarantee of yield. With the development of biological technology and the increasing demand for products, the recombinant expression of Plectasin is the preferred pathway for industrialized production. At present, Plectasin and its variant have already been expressed in prokaryotic expression system such as E coli, but it was rarely reported that E coli-based expression system was used to produce Plectasin. Problems in the prokaryotic expression system are as follows:most AMPs have remarkable activity against E. coli; most expressed proteins exist as a form of inclusion body; E. coli lacks the function of posttranslational modifications; the expressed proteins can easily be degraded by protease; In order to solve the above problems, the strategy of fusion expression is advised. The fusion expression of proteins composed of different molecular chaperones and AMPs could reduce the toxicity against the host cell and increase its solubility and stability. And specific cracking site is designed for the extraction of AMPs.With the rapid development of biotechnology, the production of AMPs by the use of microbial fermentation can be regarded as an economic and efficient method. The research focuses on the construction of recombinant E. coli for high-level expression of Plectasin, evaluation of antibacterial activity of Plectasin in vitro and optimization on prokaryotic expression conditions. It will contribute to development of the new drug, AMPs, used for drug-resistant gram-positive bacterium.[Objective]In this study, we construct recombinant strains Origami-pET-32a(+)-PS,Origami-pE-SUMO-PS,Origami-pTWIN1-C-PS; Origami-pGEX-5x-2-PS using four kinds of molecular chaperones:ThioredoxinA (TrxA), Small Ubiquitin-related modifier (SUMO), Intein2 and Glutathione-S-transferase (GST). According to the solubility of fusion proteins, cleavage efficiency and the theoretical production of Plectasin, the high-efficient chaperones and recombinant strains are determined. Further, antibacterial activity assay of Plectasin from the E coli is performed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Plectasin are evaluated and its activity and stability under different pH and temperatures is tested. At last, three main influential factors including inducing time, concentration of inducer and inducing temperature are chosen and adopted for the orthogonal experiment at three levels, which contributes to determine the optimized conditions for prokaryotic expression system. And these works will lay the foundation for future research, application and production of Plectasin.[Method]1 Construction of prokaryotic strains for high-level expression of Plectasin.1.1 Modification of Plectasin geneAccording to Mandal’s study, the amino acid sequence of Plectasin was based on the synonymous codon bias of E. coli and optimized Plectasin gene sequence was designed. The identificated sequences of corresponding restricted endonuclease and cleavage enzyme were added both terminals of Plectasin gene.1.2 Construction and identification of recombinant plasmidsModified Plectasin genes were digested with corresponding restriction endonucleases and cloned into the corresponding vectors digested with the same enzymes. Fusion gene could be constructed with four kinds of molecular chaperone (TrxA, SUMO, Intein2 and GST) and Plectasin gene. Four kinds of fusion gene were named TrxA-PS (Plectasin) Gene, SUMO-PS Gene, Intein2-PS Gene and GST-PS Gene separately. The recombinant plasmids were transformed into E. coli strain DH5a for plasmid amplification. The successful construction of recombinant plasmids were confirmed by polymerase chain reaction (PCR), restriction endonucleases digestion and DNA sequencing.1.3 Expression of fusion proteinsThe recombinant plasmids were transformed into E. coli strain Origami (DE3) for expression of fusion proteins. IPTG was added for induction of fusion proteins. The bacterial cells were harvested by centrifugation, resuspended in phosphate buffer (PB) and then lysed by sonication in an ice water bath. The proteins in induced whole cell, non-induced whole cell, liquid supernatant and sediment were separately subjected to sodium dodecyl sulfated-polyacrylamide gel electrophoresis (SDS-PAGE). The content of total proteins in liquid supernatant was assayed by the method of Bradford Protein Assay and the proportion of the target band accounting for the total proteins in ultrasound supernatant was assayed by QuantityOne. According to the previous results, the theoretical content of fusion proteins and Plectasin were calculated.1.4 Extraction of fusion proteinsRecombinant strains were induced by IPTG The bacterial cells were harvested by centrifugation, resuspended separately and then lysed by sonication. The supernatant liquid was collected by centrifugation and filtered by 0.22μm filter.1.5 Purification and proteolytic cleavage of fusion proteinsThe collected supernatant lysate was purified by Ni2+-chelating affinity chromatography and Chitin affinity chromatography, and elution was collected by FPLC. The collected fusion proteins were cleaved by corresponding enzymes.1.6 Assays of the final productsAll the final products were assayed by SDS-PAGE.2 Antibacterial activity assay of Plectasin2.1 Purity of final product cleaved from fusion proteinThe product cleaved from fusion protein SUMO-PS was assayed by Tricine-SDS-PAGE and analysed by Moclecular Imaging System (Bio-Red Laboratories) to calculate the purity.2.2 Antibacterial activity assay of PlectasinThe antibacterial activities of purified plectasin cleaved from SUMO-Ps to MRSA, VRE and PRSP were analyzed using the paper disk method (K-B method).2.3 Evaluation of MIC and MBCAfter preparation for bacterium suspension and Plectasin,96-well plates method was utilized to evaluate MIC and MBC.2.4 Influence of pH and temperatures on the activity and stability of PlectasinThe influence of pH was tested at pH values gradient (pH 2.0,4.0,6.0,8.0,10.0) and the influence of temperature tested at temperature gradient (0,25,50,75,100℃). The antibaeterial activity and stability of Plectasin were analyzed against VRE by the K-B method.3 Orthogonal experiment on optimization expression conditions of Plectasin3.1 Synthesis of fusion expression genesAccording to Mandal’s study, the amino acid sequence of Plectasin was based on the synonymous codon bias of E. coli and optimized Plectasin gene sequence was designed. Plectasin was expressed as a fusion protein with molecular chaperone SUMO in E. coli to avoid toxicity to the host. The fusion gene were named SUMO-PS Gene.3.2 Construction and identification of recombinant plasmidsThe fusion gene SUMO-PS wss digested with corresponding restriction endonucleases and cloned into the corresponding vector digested with same enzyme. The recombinant plasmids were transformed into E. coli strain DH5a for plasmid amplification. The successful construction of recombinant plasmid was confirmed by polymerase chain reaction (PCR), restriction endonucleases digestion and DNA sequencing.3.3 Orthogonal experiment designThree main influential factors:inducing time, concentration of inducer and inducing temperature were chosen and adopted for the orthogonal experiment at three levels, which contributed to determine the optimized conditions for the prokaryotic expression system.3.4 Expression and evaluation of fusion proteinAccord to the orthogonal design, the induced expression of fusion proteinswere performed in every group, and the results were assayed by 15% SDS-PAGE. By standard SDS-PAGE method, the content of soluble fusion protein of every group could be estimated.3.5 Statitics analysis and verificationAll data were analyzed by SPSS 13.0. The statistical results could indicate the intensity order of three main influencial factors and optimized inducing temperature, inducing time and concentration of inducer.[Results]1 Construction of prokaryotic strains for high-level expression of Plectasin.1.1 Construction and identification of recombinant plasmidsFour kinds of fusion gene fragments were successfully subcloned into corresponding vectors through PCR amplification. DNA sequencing confirmed that recombinant plasmids were constructed successfully.1.2 Expression of fusion proteinsThe four kinds of fusion proteins were expressed successfully, SDS-PAGE showed that expected molecular mass (22kDa,24kDa,30kDa and 30kDa) were observed in the expression products of transformed E. coli Origami (DE3). The content of total proteins in ultrasound supernatant of four kinds of recombinant strains (Origami-pET-32a(+)-PS, Origami-pE-SUMO-PS, Origami-pTWINl-C-PS, Origami-pGEX-5x-2-PS) were 0.7200g/L,0.5800g/L,1.0880g/L,0.7981g/L; The percentage of fusion proteins account for the proteins of ultrasound supernatant were 24.06%,23.44%,16.04%,21.04% respectively; The content of f fusion proteins were 0.1732 g/L,0.1359g/L,0.1745g/L,0.1708g/L respectively; The theoretical yield of Plectasin were 0.0356g/L、0.0358 g/L、0.0238g/L、0.0243g/L.1.3 Purification and proteolytic cleavage of fusion proteinsThe fusion protein TrxA-PS was cleaved and SDS-PAGE showed that the target polypeptide band could be observed, but most of fusion peptide had not been cleaved completely. The fusion protein SUMO-PS was cleaved and SDS-PAGE showed that the target polypeptide band could be observed, and most of fusion peptide had not been cleaved completely. The desalted fusion protein intein2-PS was cleaved and SDS-PAGE showed that the target polypeptide band could be observed, but most of fusion peptide had not been cleaved completely1.4 Assays of the final productsThe results of SDS-PAGE showed that the special polypeptide band of Plectasin from the cleavage of fusion protein Trx-PS, SUMO-PS, Intein2-PS, GST-PS could be observed.2 Antibacterial activity assay of Plectasin2.1 Purity of final product cleaved from fusion proteinThe results of Tricine-SDS-PAGE and Moclecular Imaging System show purity of the final product was 98.3%.2.2 Antibacterial activity assay of PlectasinThe K-B method showed Plectasin had inhibition zones to clinical MRS A, PRSP, VRE. Amp had inhibition zones to PRSP and VRE, but MRSA.2.3 Evaluation of MIC and MBCThe MIC of Plectasin to MRSA 15471114, MRSA 15471118, PRSP 31355, VRE were 64p,g/ml (14μmol/L),64μg/ml (14μmol/L),32μg/ml (7.3μmol/L),64μg/ml (14μmol/L) respectively and the MIC of Amp were 64p,g/ml (186μmol/L),128μg/ml (373μmol/L),16μg/ml (46μmol/L),32μg/ml (96μmol/L), respectively. The MBC of plectasin to MRSA 15471114, MRSA 15471118, PRSP 31355, VRE were 64μg/ml (14μmol/L),64μg/ml (14μmol/L),32μg/ml (7.3umol/L),64μg/ml (14μmol/L) respectively and the MBC of Amp were 64μg/m 1 (186μmol/L),>128μg/ml (373μmol/L),16μg/ml (46μmol/L),32μg/ml (96μmol/L) respectively. There were statistically significant differences between the MIC and MBC of plectasin and Amp for the tested clinical drug resistant strains (P<0.05).2.4 Influence of pH and temperatures on the activity of recombinant plectasinUnder different pH (2.0,4.0,6.0,8.0,10.0) and temperatures (0,25,50,75, 100℃), Plectasin showed strong antibacterial activities and stability.3 Orthogonal experiment on optimization expression conditions of Plectasin3.1 Synthesis of fusion expression geneThe fusion gene SUMO-PS fragment was successfully subcloned into the corresponding vector through PCR amplification. DNA sequencing confirmed that the recombinant plasmid was constructed successfully.3.2 Expression of fusion proteinsThe results of SDS-PAGE of the induced whole cell showed that special polypeptide band at 24kDa in every group, which indicated successful expression of fusion protein. The results of SDS-PAGE of ultrasound supernatant showed that special polypeptide band at 24kDa in every group, and the content of target protein in every group was different.3.3 Evaluation of fusion proteinsBy comparing electrophoresis figure of every group with the standard’figure, the content of soluble fusion protein could be estimated. The results showed that the content of fusion protein in group 4 was the highest (142mg/L), group 6 was least (4.71mg/L), and the content of group 5 (42.67mg/L), group 8 (44mg/L) and group 9 (46.67mg/L) were less than that of group 4.3.4 Statistics analysisThe results of statistical analysis indicated that the infuencial factors were inducing temperature, inducing time and concentration of inducer according to the intensity. The optimized inducing temperature was 30℃, the optimized inducing time 6h and the optimized concentration of IPTG 0.05mmol/L. [Conclusion]1 In this study, prokaryotic strains (Origami-pE-SUMO-PS and Origami-pET-32a(+)-PS) with high-efficient expression of Plectasin are constructed successfully. The SUMO and TrxA are appropriate chaperones used for constructing recombinant strains according to the solubility of fusion protein, cleavage efficiency and the theoretical yield of Pletasin.2 Plectasin extracted from recombinant strain Origami-pE-SUMO-PS shows strong antibacterial activity to clinical MRSA, PRSP and VRE. Under different pH and temperatures, Plecatsin shows strong antibacterial activity and stability.3 The factors are inducing temperature, inducing time and concentration of inducer according to the intensity of influence to fusion protein expression. The optimized inducing temperature was 30℃, the optimized inducing time 6h and the optimized concentration of IPTG 0.05mmol/L. |
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