融合蛋白连接肽的研究进展

融合蛋白是将两个或多个基因的编码区首尾连接,由同一调控序列控制构成的基因表达产物。对于有连接肽的融合蛋白,连接肽的设计在其中起着决定性的作用。该文从连接肽的设计、构建、分类、功能以及用连接肽构建融合蛋白时出现的问题和问题的解决等几个方面作了综述。     

分子酶工程学研究进展

酶工程的研究已经发展到分子水平,通过基因操作,已实现了许多酶的克隆和表达。定点突变成为研究酶结构与功能的常规手段,并被广泛用于改善酶的性能。体外分子进化方法则大幅提高了酶分子的进化效率,并有可能发展新功能酶。融合蛋白技术的发展使构建新型多功能融合酶成为可能。这里对分子酶工程学的研究与发展情况进行了综述。      

融合双亲短肽提高腈水合酶的热稳定性研究

腈水合酶(Nitirle hydratase, NHase)催化腈类物质转化为酰胺类物质,目前用于工业生产丙烯酰胺。但在催化过程中释放的热量易导致酶分子失活。研究通过蛋白质融合技术对腈水合酶进行分子改造,提高热稳定性。将2种双亲自组装肽（self-assembling peptides, SAPs）EAK16和ELK16分别融合至恶臭假单胞菌Pseudomonas putida NRRL-18668来源NHase非催化亚基β的N末端,构建出2种融合型NHase：EAK16-NHase和ELK16-NHase。经过表达、纯化后测定酶活力,发现EAK16-NHase和ELK16 NHase的酶活力分别为(426±14) U/mg和(372±12) U/mg,保留野生型酶活力的97%和85%。在50 ℃条件下孵育0~60 min,每5 min取样后测定残存酶活力,EAK16-NHase和ELK16-NHase酶活力半衰期（T₅₀）分别为35 min和40 min,野生型NHase为20 min。说明融合EAK16和ELK16均能提高NHase的热稳定性。研究表明融合SAPs能在不显著影响酶活力的条件下提高酶的热稳定性。     

分选酶的研究进展及其在生物技术中的应用

分选酶(sortase)普遍存在于革兰氏阳性细菌中,是一类膜结合的转肽酶,负责将表面蛋白共价结合到细胞壁的肽聚糖上。由于其独特的作用机制,分选酶在生物技术领域具有广阔的应用前景,可应用于革兰氏阳性菌的表面展示、蛋白质工程等。     

融合酶构建技术在酶的改性以及多功能酶的构建方面的应用

融合酶技术是酶的改造技术之一。应用融合酶技术还可以创造出多功能的新酶,这些新酶有望应用于食品、化工等领域。目前研究表明,融合酶在低聚糖制备,生物燃料,生物材料,氨基酸发酵以及生物传感器等领域极具应用前景。融合酶的构建技术有理性设计和非理性设计,这两种技术各有利弊。整理了近年融合酶在以上领域中的研究成果,对融合酶的工业应用进行讨论。     

天冬氨酸酶E.coli融合表达研究

以大肠杆菌基因组DNA为模板,设计引物扩增得到天冬氨酸酶基因,将其重组于胞内融合表达型T载体中,重组质粒转化表达宿主大肠杆菌BL21(DE3)。SDS-PAGE分析表明,工程菌经IPTG诱导,表达大量表观分子量约75kD的融合蛋白。经试验,工程菌细胞具有较高的天冬氨酸酶活性,融合形式的酶最适温度37℃,最适pH8.5,融合伴侣DsbA的存在对酶活没有影响。     

融合自组装双亲短肽对重组过氧化氢酶酶学性质的影响

【目的】利用融合自组装双亲短肽策略对源自枯草芽孢杆菌(Bacillus subtilis)的过氧化氢酶Kat A进行改性,以强化重组过氧化氢酶在工业中的应用适应性。【方法】将自组装双亲短肽S1vw通过连接肽PT-linker融合在Kat A的N端,构建重组质粒p HT254-S1vw-PT-kat A,将其与携带天然酶基因的p HT254-kat A分别转入枯草芽孢杆菌WB800N中进行分泌表达,之后将分离纯化得到的纯酶进行酶学性质研究。【结果】成功构建出工程菌并将胞外粗酶液通过乙醇沉淀、DEAE阴离子交换层析、疏水层析和凝胶过滤层析4步纯化,最终获得电泳纯的重组酶蛋白。酶学性质研究结果显示,融合酶S1vw-PT-Kat A和天然酶Kat A的最适反应温度均为30°C,最适反应p H值均为11.0。然而,融合酶在p H 12.0下孵育30 min的相对酶活为77.3%,是相同处理条件下天然酶相对酶活的14.9倍,在65°C和70°C下孵育30 min的相对酶活分别为19.8%和17.5%,是相同处理条件下天然酶相对酶活的1.8倍和1.7倍。此外,融合酶在4°C储存14 d后相对酶活为8...     

酶的开发利用与酶工程

近几十年来,由于酶的不断发展,酶的开发利用技术日趋成熟,形成了一门新的技术学科－酶工程．本文对酶工程所涉及的技术范围、研究内容、现状、发展趋向特别是酶的开发利用进行了较为全面的综述。     

重组单链抗体-低分子质量尿激酶融合蛋白在 CHO 细胞中的抗降解研究

抗人纤维蛋白单链抗体-低分子质量尿激酶(Ⅱn-UK)融合蛋白,兼有单链抗体对纤维蛋白的亲和性和尿激酶的溶栓活性,有望开发成为新型导向溶栓药物.但基于通用连接肽(G4S)3的Ⅱn-linker-UK融合蛋白在CHO细胞中表达时出现明显的降解.为了解决此问题,利用分子生物学方法,对Hn-UK融合蛋白进行了分子改造,包括置换连接肽,改变两个半分子(moiety)的相对位置,以及对连接肽附近明确的蛋白酶位点进行突变等方法,并分别研究了改造后的11种Ⅱn-1inker-UK或UK-linker-Ⅱn突变体在CHO细胞中分泌性表达时的稳定性,最终筛选到一种抗降解的突变体.     

有机溶剂中的酶催化——水和有机溶剂的影响

有机相酶催化是酶工程研究最活跃的领域之一,本文主要综述水及有机溶剂对有机相酶催化过程中酶的活性,稳定性,底物特异性,酶反应速率等的影响。     

工业蛋白质理性设计与应用

作为合成生物学与绿色生物制造等领域的底层核心技术,蛋白理性设计可有效解决天然功能元件性能不足等共性挑战,创制高性能人工酶元件。值此天津工业生物研究所(Tianjin Institute of Industrial Biotechnology, TIB)创立10周年之际,文中回顾了研究所在工业蛋白理性设计领域的系列重要工作进展。从酶设计方法学研究、新酶反应设计到生物催化应用等方面进行了分析讨论,并展望了本领域未来发展方向。望借此搭建学术界和产业界与酶理性设计的桥梁,促进新技术、新策略的开发应用,加速融合人工酶的基础研究与产业应用,推动我国生物制造领域的科技创新升级。     

抗菌肽融合表达研究进展

抗菌肽抗菌谱广、活性稳定,且具有与抗生素不同的抗菌机制,在抑杀病原微生物的同时不易产生耐药性,因而在食品、饲料、医药等领域具有重要的应用价值。基因工程技术是降低抗菌肽生产成本的主要方式,其中融合表达在提高抗菌肽产量方面起到了重要作用。文中综述了抗菌肽融合表达的国内外研究进展,探讨了部分融合标签用于抗菌肽表达的策略,并对今后的发展提出了自己的看法。     

大数据时代杂合酶的设计及其新趋势

酶分子的高效性和稳定性是工业广泛应用的物质基础。利用分子生物学技术可以将不同酶分子通过串联、插入、翻译后融合等方式构建成符合工业需求的杂合酶,但应用中多结构域杂合酶在表达量与酶活等方面仍存在弊端,而基于特定蛋白质结构域的多功能设计成为新趋势。高通量测序技术的发展,使得生物学家正面临着爆炸式增长的大数据集。近年来"蛋白质功能区"概念的提出,拓宽了人们对蛋白质结构与功能组织层次的认知,功能区残基聚簇的协同演化可导致同一家族不同蛋白质功能的差异。基于海量大数据分析可以快速定位特定功能区以及协同进化的关键位点,再利用合成生物学技术就可实现多种功能残基在同一蛋白质中的精准嫁接,完成天然酶分子的再设计。这将是杂合酶技术发展的新阶段,也会成为生物大数据时代下蛋白质设计的新趋势。     

Directed enzyme evolution

Laboratory evolutionists continue to generate better enzymes for industrial and research applications. Exciting developments include new biocatalysts for enantioselective carbon-carbon bond formation and fatty acid production in plants. Creative contributions to the repertoire of evolutionary methods will ensure further growth in applications and expand the scope and complexity of biological design problems that can be addressed. Researchers are also starting to elucidate mechanisms of enzyme adaptation and natural evolution by testing evolutionary scenarios in the laboratory.     

Recent advances in structure-based enzyme engineering for functional reconstruction

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidation of the structure–function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information.     

Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.     

Rational design of thiolase substrate specificity for metabolic engineering applications

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3‐hydroxyacid (3HA) pathway, also known as the reverse β‐oxidation or coenzyme‐A‐dependent chain‐elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model‐guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3‐hydroxy‐hexanoic acid and 3‐hydroxybutyric acid. We identify thiolase mutants that result in nearly 10‐fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence–structure–function relationship for this important class of enzymes.     

分子酶工程的研究进展

随着基因工程和蛋白质工程的进展和应用,酶工程在分子水平上的研究与应用也得到了迅猛发展。本着重介绍了酶基因克隆与异源表达、酶分子的定向改造和进化、融合蛋白与融合酶、酶的人工模拟(抗体酶、分子印迹技术)和端粒酶,综述了分子酶工程的研究进展、趋势及其应用。     

无细胞蛋白表达系统新进展及在生物制药工程中的应用

无细胞蛋白表达体系是一种以细胞抽提物为基础的体外合成蛋白质表达技术,具有遗传背景简单、反应操控简便等特点,已成为研究生物反应系统的重要技术手段。在研究人员的不断努力下,反应体系从原核扩展到真核蛋白质合成体系,而且目标蛋白表达量从毫克级提高到数克级每升,成本不断降低,反应规模可达到百公升级。近年来,无细胞蛋白表达系统在复杂蛋白、毒性蛋白和膜蛋白表达方面的优势逐渐体现,展示了其在生物制药领域的重要应用潜力。总之,无细胞技术已经成为异源蛋白质高效合成和生物制药领域中有巨大潜力的新策略。     

Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced beta-glucanase and xylanase expressed in Escherichia coli

A chimeric gene, Glu-Xyl, encoding Bacillus amyloliquefaciens glucanase (Glu, 24.4 kDa) and Bacillus subtilis xylanase (Xyl, 21.2 kDa), was constructed via end-to-end fusion and expressed successfully in Escherichia coli. The purified fusion protein (46.1 kDa) exhibited both glucanase and xylanase activities. Compared with parental enzymes, the Glu moiety was characterized by kinetic parameters of decreased K(m) (0.66-fold) and increased K(cat) (2.75-fold), whereas the Xyl moiety had an increased K(m) (1.37-fold) and decreased K(cat) (0.79-fold). These indicate a 3.15-fold net increase and a 31% decrease in catalytic efficiency (K(cat)/K(m)) of the Glu and Xyl moieties. Activities and stabilities of both moieties at 40-90 degrees C or pH 3.0-10.0 were compared with those of the parental enzymes. Despite some variations, common optima were 40 degrees C and pH 9.0 for the Glu moiety and parent, and 50-60 degrees C and pH 9.0 for the Xyl counterparts. Thus, the fusion enzyme Glu-Xyl was bifunctional, with greatly enhanced glucanase activity associated with a decrease in xylanase activity.