禾谷镰孢菌Fusarium graminearum α、β_2-微管蛋白的原核表达、体外聚合及药物结合研究
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摘要
禾谷镰孢菌(Fusarium graminearum)在生产中主要引起小麦赤霉病、玉米茎基腐病和穗腐病及水稻穗腐病,它不但造成作物产量损失,而且该菌产生的毒素对作物的品质造成严重的降低。主要由禾谷镰孢菌引起的小麦赤霉病是小麦生产中的一种毁灭性病害,苯并咪唑类杀菌剂如多菌灵是防治小麦赤霉有效的药剂。当前,禾谷镰孢菌已对多菌灵产生了严重的抗药性,小麦赤霉病发生有愈发严重的趋势,生产中急需防治小麦赤霉病的多菌灵替换药剂。通过遗传转化及基因定点突变技术,本实验室已证明了禾谷镰孢菌对多菌灵的抗药性是由其p2-微管蛋白基因突变引起,其突变位点包括F167Y、E198K或E198L、F200Y,50位点突变体也可产生对多菌灵的低抗。但尚未有从蛋白质水平上研究β2-微管蛋白氨基酸突变与多菌灵抗性关系的报道。
微管由微管蛋白(tubulin)和微管相关蛋白(Microtubule associated proteins, MAPs)组成,其中α、p-微管蛋白聚合形成的异源二聚体,是微管的基本单位。α、p-微管蛋白处于一个聚合/解聚的动态过程,多种以微管蛋白为靶标的药物都是同微管蛋白结合后抑制了微管的动态过程而发挥药理作用的。目前已开发出基于微管蛋白体外聚合为靶标的抗肿瘤药物筛选模型,但丝状真菌的微管蛋白还没有在离体条件下用作杀菌剂创制及筛选的靶标。
为了研究禾谷镰孢菌α、β-微管蛋白的体外聚合反应并以此开发出杀菌剂的离体筛选模型,以及在蛋白水平上理解禾谷镰孢菌对多菌灵的抗药性实质,本文在大肠杆菌中表达了禾谷镰孢菌α、β-微管蛋白,并进行了体外聚合及药剂结合研究,结果如下：
以禾谷镰孢菌cDNA为模板,PCR扩增出α1、α2-微管蛋白基因,将其克隆到pET30a+表达载体上,转化到宿主菌Rossatta (DE3) pLysS,筛选阳性克隆,进行蛋白诱导表达。SDS-PAGE及Western-blot结果表明：融合蛋白主要以包涵体形式存在,分子量约为52.1kD和55.9kD；融合蛋白能与抗His-tag的单抗发生特异性反应。通过对包涵体洗涤及透析复性后采用HisTrapTMHP Columns对融合蛋白进行纯化,可以得到纯度较高的微管蛋白。
以构建好的(pET32a-β2-tubulin)重组质粒为模板,PCR扩增出p2-微管蛋白基因,将其克隆到pET30a+表达载体上,并转化到宿主菌BL21(DE3)及Rossatta (DE3) pLysS,筛选阳性克隆,进行蛋白诱导表达,并筛选蛋白可溶性表达的诱导因子；对纯化后的蛋白进行SDS-PAGE和Western Blot验证。最终获得了在大肠杆菌中表达效率高的阳性克隆；通过对诱导温度、诱导物浓度、诱导时间、初始诱导时的菌液浓度、培养液、诱导物种类及宿主菌等七因子的综合分析,获得了β2-微管蛋白可溶性表达的诱导条件；利用HisTrapTM HP Columns对p2-微管蛋白进行纯化,可获得纯度很高的可溶性β2-微管蛋白。SDS-PAGE确认表达出的融合蛋白分子量约为51.6kD; Western-blot证实表达的融合蛋白能与His-tag及p-微管蛋白的单克隆抗体发生特异性反应。
通过改变诱导条件获得禾谷镰孢菌可溶性的α-微管蛋白,包括降低诱导温度和诱导物浓度,延长诱导时间。诱导条件改变对a-微管蛋白总量无影响,但可使包涵体中的蛋白量下降而上清中可溶性的蛋白量增加,其中上清中a1-微管蛋白的增加量较α2-微管蛋白多,说明α1-微管蛋白在大肠杆菌里更容易可溶性表达。利用HisTrapTM HP Columns对融合蛋白进行纯化,可获得纯度很高的可溶性a-微管蛋白；Western-blot证实表达的融合蛋白能与His-tag及α-微管蛋白的单克隆抗体发生特异性反应,说明α-微管蛋白在提取及纯化过程中始终保持着结构上的完整性。
研究了禾谷镰孢菌微管蛋白的体外聚合反应。微管蛋白纯化后经过透析、浓缩,可以在体外无微管相关蛋白下发生聚合反应。聚合的条件是：反应在Pipes buffer中进行,微管蛋白浓度1mg-mL"1, GTP浓度2mmol·L-1;反应温度37。C,反应时间60m,DMSO对聚合无影响；聚合在96孔酶标板上进行,在酶标仪350nm处测定反应60m之内的吸光度,每隔12s读数一次。结果表明：α2、β2-微管蛋白的聚合程度比αl、β2-微管蛋白聚合程度强；多菌灵加入可显著地抑制α1、p2或α2、β2-微管蛋白的聚合,但对已发生聚合的微管无作用；多菌灵对微管蛋白体外聚合的抑制同其活体条件下对出发菌株的抑菌效果相同。几类药物对α、β2-微管蛋白聚合的影响不同,同其活体抑菌活性也不完全一致,表明禾谷镰孢菌α1、β2-微管蛋白的体外聚合反应可用在药物的初步筛选中。
在大肠杆菌中利用pET32a+成功表达了禾谷镰孢菌不同抗性菌株的p2-微管蛋白,改变诱导条件,使β2-微管蛋白得到了可溶性表达并进行了自然条件下的纯化。不同位点的氨基酸突变对融合蛋白表达总量及可溶性蛋白表达量无明显差异。纯化的β2-微管蛋白对His-tag及β-微管蛋白单克隆抗体均有反应,说明蛋白在提取及纯化过程中一直保持着结构完整性。尽管pET32a+表达出的融合蛋白N端有大约17.3kD的标签蛋白,但同al-微管蛋白体外聚合特性与pET30a+表达的融合蛋白无差异,多菌灵对聚合的影响也相同,说明N端的标签蛋白对β2-微管蛋白与α1-微管蛋白体外聚合无影响。不同抗性菌株β2-微管蛋白与α1-微管蛋白的体外聚合存在着显著差异,198位突变导致聚合程度加强；但多菌灵对抗性菌株p2-微管蛋白与αl-微管蛋白的体外聚合无影响,表明β2-微管蛋白发生点突变后与多菌灵的亲和力降低。
采用荧光淬灭的方法研究了禾谷镰孢菌p-微管蛋白及不同抗性菌株p2-微管蛋白同多菌灵的体外亲和性。多菌灵对所有的融合蛋白都具有荧光淬灭效应,对敏感菌株β2-微管蛋白的荧光淬灭率最高,对双位点突变菌株(52-7)p2-微管蛋白荧光淬灭率最低,但对中抗菌株p2-微管蛋白的淬灭率低于高抗菌株JT04和点突变体E198K。药剂平衡试验表明,微管蛋白浓度2μmol·L-1,多菌灵浓度在20μmol·L-1,时对融合蛋白的荧光淬灭率达到最大值,故选取该浓度进行药剂动力学研究。结果表明：β2-微管蛋白与多菌灵的体外结合速率和出发菌株对多菌灵的抗性水平呈负相关,抗性水平高的菌株有较低的结合速率Kon；而解离速率Koff则同抗性水平呈正相关,抗性水平高解离速率大；亲和常数Ka同多菌灵抗性水平呈负相关,敏感菌株的p2-微管蛋白与多菌灵的亲和常数大于抗性菌株的。p1-微管蛋白与多菌灵的亲和常数Ka介于敏感菌株p2-微管蛋白和低抗点突变体Y50C p2-微管蛋白之间,表明其在禾谷镰孢菌对多菌灵抗性表型中也有一定作用。
Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley because it reduces grain yield and quality and results in the contamination of grain with mycotoxins. Benzimidazole fungicides, including carbendazim (methyl benzimidazol-2-yl carbendazim, MBC) and thiophanate, have been relied to control the disease for thirty years. However, the appearance of resistance has hindered the continuous use of the fungicides, which causes severe prevalence of the disease. Because of the appearance of resistance, there is a current need to screen and develop new compound to control FHB. The mechanism of F. graminearum to MBC has been studied by genetic transformation and site-directed mutagenesis. The results have showed that site mutation on β2-tubulin could cause resistance of different levels, including F167Y, E198K, E198L and F200Y. There is no report of resistant mechanism on the protein level up yet now.
Microtubules consist of repeating α/β tubulin heterodimers, and because of tubulin's GTPase activity, they are highly dynamic. Microtubule dynamics can be inhibited by many antimitotic agents, and such inhibition is an important mode of action of anti-tumor drugs, antiprotozoal compounds and fungicides. Because of the success of tubulin-binding agents in anti-tumor drug screening, there is continuing interest in discovering new substances that interfere with microtubule-mediated processes. Though tubulins have been used as the target to screen and develop anti-tumor agents and anti-leishmanial drugs, fungal tubulins have not been used to screen for fungicides.
In research concerning the development of an screening model for detection of anti-fungus compounds based on α/β tubulin assembly in vitro, and studying the resistant mechanism on protein level, the α-and β-tubulins of F. graminearum have been cloned on the pET vectors and expressed in Escherichia coli, and the polymerization in vitro and binding kinetics with carbendazim were also studied. The results are as follows.
a-tubulin genes were amplified from F. graminearum cDNA and cloned to the vector pET30a+, then transformed into the host Rossatta (DE3) pLysS. After the positive clones were screened by the colony PCR and double digestion, the induced fusion proteins were obtained and verified by SDS-PAGE and Western blot. The positive clones which could express more fusion protein were screened, however, the fusion proteins formed mainly inclusion bodies. The molecular weight of fusion proteins were confirmed to be52.1kD and55.9kD by SDS-PAGE, which also showed specific activity to anti-6xHis monoclonal antibody. After washing of inclusion bodies using buffer containing2and3mol/L urea, the purity of fusion protein would increase. The soluble fusion protein was obtained by dialysis to binding buffer and then a-tubulins were purified by HisTrapTM HP Columns. The purified tubulins can be used in the studies of new fungicide screening in vitro that target tubulin.
β2-tubulin of F. graminearum was expressed in E. coli in soluble form and purified by HisTrapTM HP Columns. β2-tubulin gene contained in the plasmid,(pET32a+-β2-tubulin) was amplified, cloned to the vector pET30a+and, then transformed into the hosts:Rossatta (DE3) pLysS and BL21(DE3). After the positive clones were screened by the colony PCR and double enzymatic digestion, the induced fusion proteins were obtained and verified by the SDS-PAGE and Western blot. In order to express the fusion protein in soluble form, the inducing factors, including temperature, induction time, IPTG (Isopropyl P-D-Thiogalactoside) concentration, cell density, medium composition and hosts, were screened. The positive clones which could express more fusion protein after induced were screened, however, the fusion proteins formed inclusion bodies. The molecular weight of fusion proteins were confirmed to be52kD by SDS-PAGE, which also showed specific activity to anti-6×His monoclonal antibody. After the optimization of imidazole concentration in binding and wash buffer, the soluble fusion protein was purified and its structural integrity was preserved through the purification process by the verification of western blot. The methods described here can be used to express and purify other recombinant proteins in soluble form in E. coli. The purified fusion tubulin can be used in the studies of tubulin-target drug resistant mechanisms as well as high throughout screening of new fungicide.
The expressed a-tubulins existed in both soluble and insoluble forms, and the expression of soluble form was affected by isopropyl-β-D-thiogalactoside (IPTG) concentration, incubation temperature, incubation time, and culture density. The soluble proteins, which were purified by Ni2+affinity chromatography, were recognized by His-tag and a-tubulin antibody. The structural integrity of a-tubulins had been preserved throughout the expression and purification processes and the a-tubulins were suitable for high-throughout screening of candidate fungicides.
After purified, dialyzed and concentrated, F. graminearum tubulins were polymerized. It was the first report that tubulins of F. graminearum polymerized in the absence of microtubule-associated proteins (MAPs). Polymerization was assayed in the PIPES buffer, where the concentration of tubulin and GTP was1mg-mL-1and2mmo1·L-1respectively. GTP can initiate the polymerization at37℃. Tubulin polymerization was assayed turbidimetrically at350nm for60minutes with an12seconds interval on96-well plates, using a Molecular Devices VersaMax microplate reader equipped with temperature controllers. There was no effect of DMSO (Dimethyl sulfoxide) on polymerization. The results showed that the assembly of of a2/β2-tubulin was higher than that of α1/β2-tubulin and the absorbance did not increase at350nm in the presence of carbendazim, which was consistent with sensitivity of the starting strain2021to this fungicide. Carbendazim had no effect on polymerized tubulins mixture and the absorbance did not decrease, however. There were different inhibition types of pesticides on the polymerization, which were not entirely consistent with their antifungal activity in vitro. The screening model based on the polymerization assay of F. graminearum α/β2tubulin can be used on the primary screening of new compound which targets tubulin.
β2-tubulins originated from the resistant strains and site-directed mutants (SDMs)were cloned into pET32a and expressed in soluble form in E. coli after optimization of induction factors. The soluble β2-tubulins were purified and there were no differences of total fusion protein and soluble forms of amino acid substitution on β2-tubulin. The purified β2-tubulins could recognize the anti-His-tag and anti-β-tubulin monoclonal antibody, indicating that the structural integrity of β2-tubulins had been preserved throughout the expression and purification processes. Polymerization degree between α1-tubulin and β2-tubulins expressed by pET32a was consistent with that with P2-tubulin expressed by pET30a, so the unwanted amino acids on N terminals of P2-tubulins, including Thioredoxin, His-tag, S-tag, had no effect on polymerization. Mutation on β2-tubulin could promote assembly, where mutation on site198had the most dramatic ability. Carbendazim had no effect on polymerization in vitro between β2-tubulins of SDMs and a1-tubulin, implying that the binding force between carbendazim and β2-tubulins of SDMs was weaker than β2-tubulin of wild type strain which was sensitive to carbendazim.
The in vitro binding characteristics of carbendazim with β-tubulins of different mutation sites, including Y50C, F167Y, E198K, E198L, F200Y and two mutation sites,(G17S+F167Y), were studied by fluorescence quenching. Fluorescence of all these recombinant tubulins was quenched following the addition of carbendazim, in which the β2-tubulin of wild sensitive strain had the highest quenching rate and the β2-tubulin with two mutation sites has the lowest, but the quenching rate of moderately resistant strains were higher than that of highly resistant ones. In the equilibrium binding studies, carbendazim with20μmol·L-1concentration could achieve the highest quenching rate for all recombinant tubulins, so the concentration was used in the binding kinetics experiment. The apparent association constant Ka)of β2-tubulin of site-directed mutants was negatively correlated with the resistant level of starting strain to carbendazim. However, the dissociation rates (Koff) was positively correlated with the resistant level. So that was affinity constant (Ka), which mean that the strain with high resistant level had the large Ka value. The Ka value of β1-tubulin to carbendazim was intermediate between β2-tubulin of wild sensitive strain and low resistance, implying that β1-tubulin contributed to the carbendazim resistance of F. graminearum.
微管由微管蛋白(tubulin)和微管相关蛋白(Microtubule associated proteins, MAPs)组成,其中α、p-微管蛋白聚合形成的异源二聚体,是微管的基本单位。α、p-微管蛋白处于一个聚合/解聚的动态过程,多种以微管蛋白为靶标的药物都是同微管蛋白结合后抑制了微管的动态过程而发挥药理作用的。目前已开发出基于微管蛋白体外聚合为靶标的抗肿瘤药物筛选模型,但丝状真菌的微管蛋白还没有在离体条件下用作杀菌剂创制及筛选的靶标。
为了研究禾谷镰孢菌α、β-微管蛋白的体外聚合反应并以此开发出杀菌剂的离体筛选模型,以及在蛋白水平上理解禾谷镰孢菌对多菌灵的抗药性实质,本文在大肠杆菌中表达了禾谷镰孢菌α、β-微管蛋白,并进行了体外聚合及药剂结合研究,结果如下：
以禾谷镰孢菌cDNA为模板,PCR扩增出α1、α2-微管蛋白基因,将其克隆到pET30a+表达载体上,转化到宿主菌Rossatta (DE3) pLysS,筛选阳性克隆,进行蛋白诱导表达。SDS-PAGE及Western-blot结果表明：融合蛋白主要以包涵体形式存在,分子量约为52.1kD和55.9kD；融合蛋白能与抗His-tag的单抗发生特异性反应。通过对包涵体洗涤及透析复性后采用HisTrapTMHP Columns对融合蛋白进行纯化,可以得到纯度较高的微管蛋白。
以构建好的(pET32a-β2-tubulin)重组质粒为模板,PCR扩增出p2-微管蛋白基因,将其克隆到pET30a+表达载体上,并转化到宿主菌BL21(DE3)及Rossatta (DE3) pLysS,筛选阳性克隆,进行蛋白诱导表达,并筛选蛋白可溶性表达的诱导因子；对纯化后的蛋白进行SDS-PAGE和Western Blot验证。最终获得了在大肠杆菌中表达效率高的阳性克隆；通过对诱导温度、诱导物浓度、诱导时间、初始诱导时的菌液浓度、培养液、诱导物种类及宿主菌等七因子的综合分析,获得了β2-微管蛋白可溶性表达的诱导条件；利用HisTrapTM HP Columns对p2-微管蛋白进行纯化,可获得纯度很高的可溶性β2-微管蛋白。SDS-PAGE确认表达出的融合蛋白分子量约为51.6kD; Western-blot证实表达的融合蛋白能与His-tag及p-微管蛋白的单克隆抗体发生特异性反应。
通过改变诱导条件获得禾谷镰孢菌可溶性的α-微管蛋白,包括降低诱导温度和诱导物浓度,延长诱导时间。诱导条件改变对a-微管蛋白总量无影响,但可使包涵体中的蛋白量下降而上清中可溶性的蛋白量增加,其中上清中a1-微管蛋白的增加量较α2-微管蛋白多,说明α1-微管蛋白在大肠杆菌里更容易可溶性表达。利用HisTrapTM HP Columns对融合蛋白进行纯化,可获得纯度很高的可溶性a-微管蛋白；Western-blot证实表达的融合蛋白能与His-tag及α-微管蛋白的单克隆抗体发生特异性反应,说明α-微管蛋白在提取及纯化过程中始终保持着结构上的完整性。
研究了禾谷镰孢菌微管蛋白的体外聚合反应。微管蛋白纯化后经过透析、浓缩,可以在体外无微管相关蛋白下发生聚合反应。聚合的条件是：反应在Pipes buffer中进行,微管蛋白浓度1mg-mL"1, GTP浓度2mmol·L-1;反应温度37。C,反应时间60m,DMSO对聚合无影响；聚合在96孔酶标板上进行,在酶标仪350nm处测定反应60m之内的吸光度,每隔12s读数一次。结果表明：α2、β2-微管蛋白的聚合程度比αl、β2-微管蛋白聚合程度强；多菌灵加入可显著地抑制α1、p2或α2、β2-微管蛋白的聚合,但对已发生聚合的微管无作用；多菌灵对微管蛋白体外聚合的抑制同其活体条件下对出发菌株的抑菌效果相同。几类药物对α、β2-微管蛋白聚合的影响不同,同其活体抑菌活性也不完全一致,表明禾谷镰孢菌α1、β2-微管蛋白的体外聚合反应可用在药物的初步筛选中。
在大肠杆菌中利用pET32a+成功表达了禾谷镰孢菌不同抗性菌株的p2-微管蛋白,改变诱导条件,使β2-微管蛋白得到了可溶性表达并进行了自然条件下的纯化。不同位点的氨基酸突变对融合蛋白表达总量及可溶性蛋白表达量无明显差异。纯化的β2-微管蛋白对His-tag及β-微管蛋白单克隆抗体均有反应,说明蛋白在提取及纯化过程中一直保持着结构完整性。尽管pET32a+表达出的融合蛋白N端有大约17.3kD的标签蛋白,但同al-微管蛋白体外聚合特性与pET30a+表达的融合蛋白无差异,多菌灵对聚合的影响也相同,说明N端的标签蛋白对β2-微管蛋白与α1-微管蛋白体外聚合无影响。不同抗性菌株β2-微管蛋白与α1-微管蛋白的体外聚合存在着显著差异,198位突变导致聚合程度加强；但多菌灵对抗性菌株p2-微管蛋白与αl-微管蛋白的体外聚合无影响,表明β2-微管蛋白发生点突变后与多菌灵的亲和力降低。
采用荧光淬灭的方法研究了禾谷镰孢菌p-微管蛋白及不同抗性菌株p2-微管蛋白同多菌灵的体外亲和性。多菌灵对所有的融合蛋白都具有荧光淬灭效应,对敏感菌株β2-微管蛋白的荧光淬灭率最高,对双位点突变菌株(52-7)p2-微管蛋白荧光淬灭率最低,但对中抗菌株p2-微管蛋白的淬灭率低于高抗菌株JT04和点突变体E198K。药剂平衡试验表明,微管蛋白浓度2μmol·L-1,多菌灵浓度在20μmol·L-1,时对融合蛋白的荧光淬灭率达到最大值,故选取该浓度进行药剂动力学研究。结果表明：β2-微管蛋白与多菌灵的体外结合速率和出发菌株对多菌灵的抗性水平呈负相关,抗性水平高的菌株有较低的结合速率Kon；而解离速率Koff则同抗性水平呈正相关,抗性水平高解离速率大；亲和常数Ka同多菌灵抗性水平呈负相关,敏感菌株的p2-微管蛋白与多菌灵的亲和常数大于抗性菌株的。p1-微管蛋白与多菌灵的亲和常数Ka介于敏感菌株p2-微管蛋白和低抗点突变体Y50C p2-微管蛋白之间,表明其在禾谷镰孢菌对多菌灵抗性表型中也有一定作用。
Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley because it reduces grain yield and quality and results in the contamination of grain with mycotoxins. Benzimidazole fungicides, including carbendazim (methyl benzimidazol-2-yl carbendazim, MBC) and thiophanate, have been relied to control the disease for thirty years. However, the appearance of resistance has hindered the continuous use of the fungicides, which causes severe prevalence of the disease. Because of the appearance of resistance, there is a current need to screen and develop new compound to control FHB. The mechanism of F. graminearum to MBC has been studied by genetic transformation and site-directed mutagenesis. The results have showed that site mutation on β2-tubulin could cause resistance of different levels, including F167Y, E198K, E198L and F200Y. There is no report of resistant mechanism on the protein level up yet now.
Microtubules consist of repeating α/β tubulin heterodimers, and because of tubulin's GTPase activity, they are highly dynamic. Microtubule dynamics can be inhibited by many antimitotic agents, and such inhibition is an important mode of action of anti-tumor drugs, antiprotozoal compounds and fungicides. Because of the success of tubulin-binding agents in anti-tumor drug screening, there is continuing interest in discovering new substances that interfere with microtubule-mediated processes. Though tubulins have been used as the target to screen and develop anti-tumor agents and anti-leishmanial drugs, fungal tubulins have not been used to screen for fungicides.
In research concerning the development of an screening model for detection of anti-fungus compounds based on α/β tubulin assembly in vitro, and studying the resistant mechanism on protein level, the α-and β-tubulins of F. graminearum have been cloned on the pET vectors and expressed in Escherichia coli, and the polymerization in vitro and binding kinetics with carbendazim were also studied. The results are as follows.
a-tubulin genes were amplified from F. graminearum cDNA and cloned to the vector pET30a+, then transformed into the host Rossatta (DE3) pLysS. After the positive clones were screened by the colony PCR and double digestion, the induced fusion proteins were obtained and verified by SDS-PAGE and Western blot. The positive clones which could express more fusion protein were screened, however, the fusion proteins formed mainly inclusion bodies. The molecular weight of fusion proteins were confirmed to be52.1kD and55.9kD by SDS-PAGE, which also showed specific activity to anti-6xHis monoclonal antibody. After washing of inclusion bodies using buffer containing2and3mol/L urea, the purity of fusion protein would increase. The soluble fusion protein was obtained by dialysis to binding buffer and then a-tubulins were purified by HisTrapTM HP Columns. The purified tubulins can be used in the studies of new fungicide screening in vitro that target tubulin.
β2-tubulin of F. graminearum was expressed in E. coli in soluble form and purified by HisTrapTM HP Columns. β2-tubulin gene contained in the plasmid,(pET32a+-β2-tubulin) was amplified, cloned to the vector pET30a+and, then transformed into the hosts:Rossatta (DE3) pLysS and BL21(DE3). After the positive clones were screened by the colony PCR and double enzymatic digestion, the induced fusion proteins were obtained and verified by the SDS-PAGE and Western blot. In order to express the fusion protein in soluble form, the inducing factors, including temperature, induction time, IPTG (Isopropyl P-D-Thiogalactoside) concentration, cell density, medium composition and hosts, were screened. The positive clones which could express more fusion protein after induced were screened, however, the fusion proteins formed inclusion bodies. The molecular weight of fusion proteins were confirmed to be52kD by SDS-PAGE, which also showed specific activity to anti-6×His monoclonal antibody. After the optimization of imidazole concentration in binding and wash buffer, the soluble fusion protein was purified and its structural integrity was preserved through the purification process by the verification of western blot. The methods described here can be used to express and purify other recombinant proteins in soluble form in E. coli. The purified fusion tubulin can be used in the studies of tubulin-target drug resistant mechanisms as well as high throughout screening of new fungicide.
The expressed a-tubulins existed in both soluble and insoluble forms, and the expression of soluble form was affected by isopropyl-β-D-thiogalactoside (IPTG) concentration, incubation temperature, incubation time, and culture density. The soluble proteins, which were purified by Ni2+affinity chromatography, were recognized by His-tag and a-tubulin antibody. The structural integrity of a-tubulins had been preserved throughout the expression and purification processes and the a-tubulins were suitable for high-throughout screening of candidate fungicides.
After purified, dialyzed and concentrated, F. graminearum tubulins were polymerized. It was the first report that tubulins of F. graminearum polymerized in the absence of microtubule-associated proteins (MAPs). Polymerization was assayed in the PIPES buffer, where the concentration of tubulin and GTP was1mg-mL-1and2mmo1·L-1respectively. GTP can initiate the polymerization at37℃. Tubulin polymerization was assayed turbidimetrically at350nm for60minutes with an12seconds interval on96-well plates, using a Molecular Devices VersaMax microplate reader equipped with temperature controllers. There was no effect of DMSO (Dimethyl sulfoxide) on polymerization. The results showed that the assembly of of a2/β2-tubulin was higher than that of α1/β2-tubulin and the absorbance did not increase at350nm in the presence of carbendazim, which was consistent with sensitivity of the starting strain2021to this fungicide. Carbendazim had no effect on polymerized tubulins mixture and the absorbance did not decrease, however. There were different inhibition types of pesticides on the polymerization, which were not entirely consistent with their antifungal activity in vitro. The screening model based on the polymerization assay of F. graminearum α/β2tubulin can be used on the primary screening of new compound which targets tubulin.
β2-tubulins originated from the resistant strains and site-directed mutants (SDMs)were cloned into pET32a and expressed in soluble form in E. coli after optimization of induction factors. The soluble β2-tubulins were purified and there were no differences of total fusion protein and soluble forms of amino acid substitution on β2-tubulin. The purified β2-tubulins could recognize the anti-His-tag and anti-β-tubulin monoclonal antibody, indicating that the structural integrity of β2-tubulins had been preserved throughout the expression and purification processes. Polymerization degree between α1-tubulin and β2-tubulins expressed by pET32a was consistent with that with P2-tubulin expressed by pET30a, so the unwanted amino acids on N terminals of P2-tubulins, including Thioredoxin, His-tag, S-tag, had no effect on polymerization. Mutation on β2-tubulin could promote assembly, where mutation on site198had the most dramatic ability. Carbendazim had no effect on polymerization in vitro between β2-tubulins of SDMs and a1-tubulin, implying that the binding force between carbendazim and β2-tubulins of SDMs was weaker than β2-tubulin of wild type strain which was sensitive to carbendazim.
The in vitro binding characteristics of carbendazim with β-tubulins of different mutation sites, including Y50C, F167Y, E198K, E198L, F200Y and two mutation sites,(G17S+F167Y), were studied by fluorescence quenching. Fluorescence of all these recombinant tubulins was quenched following the addition of carbendazim, in which the β2-tubulin of wild sensitive strain had the highest quenching rate and the β2-tubulin with two mutation sites has the lowest, but the quenching rate of moderately resistant strains were higher than that of highly resistant ones. In the equilibrium binding studies, carbendazim with20μmol·L-1concentration could achieve the highest quenching rate for all recombinant tubulins, so the concentration was used in the binding kinetics experiment. The apparent association constant Ka)of β2-tubulin of site-directed mutants was negatively correlated with the resistant level of starting strain to carbendazim. However, the dissociation rates (Koff) was positively correlated with the resistant level. So that was affinity constant (Ka), which mean that the strain with high resistant level had the large Ka value. The Ka value of β1-tubulin to carbendazim was intermediate between β2-tubulin of wild sensitive strain and low resistance, implying that β1-tubulin contributed to the carbendazim resistance of F. graminearum.
引文
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