利用ELP-Intein系统在大肠杆菌中生产抗菌肽DLP4

DLP4 （defensin-like peptide 4）是一种新型昆虫防御素抗菌肽,对革兰氏阳性细菌具有强大的抗菌活性而且不易产生抗药性。本研究利用类弹性蛋白（elastin-like polypeptide, ELP）的相变特性和蛋白质内含子（intein, I）的C端断裂系统,通过构建重组表达质粒pET-ELP-I-DLP4,以大肠杆菌（Escherichia coli）作为宿主细胞,诱导表达后的重组蛋白通过简单的离心、pH和温度转变进行纯化得到DLP4。研究中发现,在表达纯化过程中蛋白质内含子发生了C端提前断裂。为了解决这一问题,将其断裂为N端片段（I0_N）和C端片段（I0_C）后,分别与ELP或DLP4融合,构建了pET-ELP-I0_N和pET-ELP-I0_C-DLP4两种重组表达质粒。分别在大肠杆菌中诱导表达,将表达后的菌液混合,使蛋白质内含子恢复C端断裂活性,最终得到的DLP4的得率约为1.49 mg/L。抑菌试验证明纯化的DLP4表现出预期活性,这为DLP4在原核系统中的表达纯化提供...     

类弹性蛋白标签在重组蛋白分离纯化中的应用

类弹性蛋白(elastin-like polypeptide,ELP)是一种非常有前途的重组蛋白分离纯化标签.这种人工合成的蛋白质多肽是由五肽重复序列单元(VPGXG)串联组成,具有温度诱导的可逆相变特性.ELP与目的蛋白融合后,赋予重组蛋白相类似的可逆相变性质.多次可逆相变循环(inverse transition cycling,ITC)之后,就可以从蛋白溶液混合液中选择性地分离出ELP融合蛋白,再经特异性酶切或者改变环境条件引发内含肽发生自我剪切去除ELP标签,从而得到单一的目的蛋白,实现简单快速分离纯化重组蛋白.目前,该技术已成功应用于原核大肠杆菌和植物表达系统中.大肠杆菌表达ELP-绿色荧光融合蛋白的最高产量可达1.6 g/L.这种非色谱分离纯化重组蛋白的方法具有技术简单、操作快速、成本低、易于扩大等优点.重点从该技术的原理、技术路线以及发展方向进行综述.     

内含肽在构建亲和纯化系统中的应用

内含肽是前体未成熟蛋白中的一段具有自我剪接功能的多肽链,在蛋白质纯化、蛋白质连接、环肽制备、蛋白标记以及生物传感器等方面广泛应用。本文综述了内含肽应用于蛋白质亲和纯化的发展历程,分别对层析型和非层析型内含肽纯化体系进行了分析和讨论,并总结了对控制内含肽断裂反应所进行的研究,为进一步改善内含肽介导蛋白质纯化提供依据和线索。     

高剪接活性断裂蛋白质内含子的体内切割

蛋白质内含子介导的断裂(切割)反应被用于蛋白质纯化、连接和环化等,但目前仍存在断裂效率低、断裂反应的不可控、产物复杂等问题。蛋白质内含子的定点突变可导致其N端或C端断裂。其末位氨基酸突变则剪接反应第3步天冬酰胺环化无法进行,发生N端断裂;其首位氨基酸发生突变则剪接反应第一步酰基重排及其后续步骤均无法进行,而天冬酰胺环化仍可进行,发生C端断裂。利用已获得的高剪接活性的S1和S11型断裂蛋白质内含子Ssp GyrB,分别将其参与剪接反应的首位半胱氨酸或末位天冬酰胺突变为丙氨酸,构建能够发生一端断裂的断裂蛋白质内含子。研究结果表明,突变后断裂蛋白质内含子的剪接反应几乎不发生,其断裂活性有不同程度的提高,获得了在大肠杆菌体内具有较高效断裂活性的断裂蛋白质内含子。这将为进一步研究其体外可控性剪接、构建高效的蛋白纯化系统和深入研究蛋白质内含子的剪接机制提供基础。     

连接肽对融合蛋白ELP[I]_(30)-linker-eGFP相变的影响

研究G4S和Poly N连接肽对融合蛋白ELP[I]30-linker-eGFP相变的影响.将编码两种不同连接肽G4S和Poly N的绿色荧光蛋白(enhanced green fluorescent protein,eGFP)基因克隆到pET28-ELP[I]30表达载体中,在宿主菌E.coli BLR(DE3)中经IPTG诱导表达ELP[I]30-linker-eGFP,通过可逆相变循环(inverse transition cycling,ITC)及镍柱亲和层析纯化ELP[I]30-linker-eGFP蛋白.结果显示,成功构建、表达具有活性的两种连接肽的融合蛋白ELP[I]30-linker-eGFP,连接肽G4S使融合蛋白产生不可逆相变,而Poly N不影响融合蛋白可逆相变,该研究对类弹性蛋白标签的应用具有指导意义.     

PRP8蛋白质反式剪接系统的建立

真菌病原体Cryptococcus neoformansAD血清型剪接体蛋白PRP8蛋白质内含子是目前 发现的第2个存在于真核生物体核基因组中的蛋白质内含子.它的宿主基因prp8编码的PRP 8蛋白作为剪接体的1个组分,是1个高度保守的mRNA剪接蛋白.将组氨酸标签插入克隆自真菌病原体Cryptococcus neoformans AD血清型的PRP8蛋白质内含子中,并将该蛋白质内含子进行人工断裂,获得断裂蛋白质内含子,在大肠杆菌中鉴定其剪接活性.研究结果表明：所获得的改造型蛋白质内含子均表现出高效的剪接活性.利用此Cryptococcus neoformansAD血清型PRP8 断裂蛋白质内含子,成功构建了蛋白质反式剪接系统.这一反式剪接系统可用于其他蛋白质的连接与合成,有望成为蛋白质工程中的一种有用工具.     

蛋白质内含子介导的蛋白纯化系统在变性条件下的应用

由蛋白质内含子介导的亲和蛋白质纯化系统(IMPACT)已得到广泛应用,通过其纯化得到的目的蛋白不含蛋白纯化标签以及多余的氨基酸残基,且操作简单成本低廉。这些优点使得其相对于其他蛋白纯化系统有着无与伦比的优势。但是现有报道都局限于非变性条件下使用,这往往会限制其在一些包涵体蛋白变性条件下使用。以一已知表达形成包涵体形式的丝素蛋白为例,研究IMPACT系统在时变性条件下使用变性剂浓度、温度和诱导断裂还原剂浓度。实验表明,在4 M尿素,100 mM DTT室温作用下蛋白质内含子会获得最大断裂效率(80%)。柱上在线断裂实验表明,其最终蛋白得率超过65%。     

ELP[Ⅰ]50标签在重组硫氧还蛋白分离纯化中的应用及PEG对其相变温度的影响

[目的]使用自行设计的类弹性蛋白(Elastin-like protein,ELP) ELP[Ⅰ]50作为非色谱纯化标签,分离纯化重组硫氧还蛋白(Thioredoxin,Trx),并研究聚乙二醇(Polyethyleneglycol,PEG)对ELP[Ⅰ]50-Trx相变温度(Inverse temperature transition,Tt)的影响.[方法]人工合成Trx基因,将其亚克隆到自行构建的表达载体pET28编码ELP[Ⅰ]50标签下游,转入大肠杆菌BLR(DE3)进行表达.融合蛋白表达后,采用可逆相变循环(Inverse transition cycling,ITC)分离纯化,并检测不同浓度PEG时的Tt值.[结果]成功表达、分离纯化出融合蛋白ELP[Ⅰ]50-Trx,检测出该蛋白浓度为25 μmol/L时,Tt为28.6℃;而当PEG的浓度为5％、10％、15％、20％时,Tt分别降至22.3℃、15.9℃、6℃、0℃.[结论]ELP[Ⅰ]50标签高效纯化重组蛋白具有操作简便、成本较低、易于扩大的优势,而PEG能降低蛋白的Tt值,进一步增强分离纯化效果,扩大使用范围,可望应用于分离纯化多种重组蛋白.     

DNA展示系统介导的断裂蛋白质内含子体外定向进化方法的研究

蛋白质内含子对于外显子的选择性限制了它的应用范围。目前,已经建立的卡那霉素定向进化系统适用于微小蛋白质内含子,但不适用于断裂蛋白质内含子。为了研究适用于断裂蛋白质内含子的定向进化的方法,我们引入了DNA展示系统。在该系统中,生物素化基因在人工细胞中与其表达的融合蛋白（内含子C端-外显子-生物素结合蛋白）形成DNA 蛋白质连接体。只有能与后续加入的N端蛋白质内含子前体（Flag-内含子N端）发生剪接反应的DNA 蛋白质连接体,才能被加上旗帜标签（Flag）,从而被抗旗帜标签抗体纯化柱（Anti Flag antibody M2 agarose）筛选富集。为了验证该系统的可行性,实验构建了2个基因,即含有具剪接活性的蛋白质内含子的阳性基因IRC和含有不具剪接活性的蛋白质内含子的阴性基因IRCM。实验首先通过Western印迹和琼脂糖凝胶电泳证明,人工细胞中的体外转录翻译系统不仅可高表达500个氨基酸的蛋白质,而且表达的蛋白中内含子仍保持原有的剪接能力。生物素结合蛋白能结合95%的DNA,并且形成的DNA 蛋白质连接体可以被筛选富集,最终证明了该系统用于断裂蛋白质内含子定向进化筛选的可行性。随后,为了检测系统的富集效率,制备了人为的基因“突变库”,即将基因IRC和IRCM以摩尔比1:10的比例混合,经过2轮筛选后,阳性基因IRC可以被10倍富集,进一步证明了体外筛选方法的可行性。该方法为后续针对不同宿主进化出不同的断裂蛋白质内含子提供筛选方法支持,也为断裂蛋白质内含子的在生物技术和研究领域的应用奠定了基础。     

ELP[I]50标签在重组硫氧还蛋白分离纯化中的应用及PEG对其相变温度的影响

【目的】使用自行设计的类弹性蛋白(Elastin-like protein, ELP) ELP[I]50作为非色谱纯化标签, 分离纯化重组硫氧还蛋白(Thioredoxin, Trx), 并研究聚乙二醇(Polyethylene glycol, PEG)对ELP[I]50-Trx相变温度(Inverse temperature transition, Tt)的影响。【方法】人工合成Trx基因, 将其亚克隆到自行构建的表达载体pET28编码ELP[I]50标签下游, 转入大肠杆菌BLR(DE3)进行表达。融合蛋白表达后, 采用可逆相变循环(Inverse transition cycling, ITC)分离纯化, 并检测不同浓度PEG时的Tt值。【结果】成功表达、分离纯化出融合蛋白ELP[I]50-Trx, 检测出该蛋白浓度为25 μmol/L时, Tt为28.6 °C; 而当PEG的浓度为5%、10%、15%、20%时, Tt分别降至22.3 °C、15.9 °C、6 °C、0 °C。【结论】ELP[I]50标签高效纯化重组蛋白具有操作简便、成本较低、易于扩大的优势, 而PEG能降低蛋白的Tt值, 进一步增强分离纯化效果, 扩大使用范围, 可望应用于分离纯化多种重组蛋白。     

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.     

Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

A self‐cleaving elastin‐like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self‐cleaving ELP‐intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP‐intein purification. Thus, this simple ELP‐based method should be applicable for the nonchromatographic purification of other recombinant, in vivo‐assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large‐scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.     

A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification

Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein–ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.     

High-throughput purification of recombinant proteins using self-cleaving intein tags

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.     

Recombinant protein purification by self-cleaving aggregation tag

A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 degrees C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50-100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8-24 h depending on specific process parameters.     

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and β-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.     

Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli

The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate--chitin (IMPACT) system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale.     

Engineering a recyclable elastin‐like polypeptide capturing scaffold for non‐chromatographic protein purification

Previously, we reported a non‐chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin‐like polypeptide (ELP) to provide fast and cost‐effective protein purification. However, the bound dockerin‐intein tag cannot be completely dissociated from the ELP‐cohesin capturing scaffold due to the high binding affinity, resulting in a single‐use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium‐coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA‐mediated dissociation of the bound dockerin‐intein tag from the ELP‐cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non‐chromatographic based affinity method provides an attractive approach for efficient and cost‐effective protein purification. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:968–971, 2013     

Enhanced purification and characterization of the PfeIF4A (PfH45) helicase from Plasmodium Falciparum using a codon-optimised clone

Thymosin α1-thymopentin (Tα1-TP5) fusion peptide has been proved to be an immune regulator based on its higher immunoregulatory activity than Tα1 and TP5. To obtain Tα1-TP5 more effectively and economically, Tα1-TP5 was genetically fused to a self-cleaving intein-chitin binding domain tag for purification via chitin beads in Escherichia coli. After affinity purification, the target peptide was released from the chitin beads via self-cleaving intein ((INTervening protEIN) induced by dithiothreitol. Further, Tα1-TP5 was purified by Superdex 30 and identified by Tricine-SDS-PAGE and electrospray ionization-mass spectrometry. Finally, about 7.6 mg Tα1-TP5 purified from the soluble fraction and inclusion bodies was obtained from 1 L culture media. The purity was 95% after a series of chromatographic purification steps. In vitro, the purified Tα1-TP5 could stimulate the proliferation of mouse splenic lymphocytes. Overall, this work demonstrated that Tα1-TP5 was purified with low cost and high efficiency, greatly expanding its potential use as an immune regulator.