无细胞蛋白质芯片的制备及其应用

蛋白质芯片是生物技术和功能蛋白组学的关键技术之一. 传统的生产蛋白的方 法周期长且费用高. 无细胞蛋白质合成系统和蛋白芯片的结合, 避免了基因的克隆、 蛋白的表达、纯化和保存等繁琐过程, 使整个无细胞蛋白芯片的制备过程快捷、迅速 和高效. 本文详细综述了无细胞蛋白质合成系统及其分类、无细胞表达系统在制备蛋 白质芯片方面的研究进展, 并探讨了无细胞蛋白质芯片在蛋白组学研究中的最新应用.     

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。     

乳酸菌蛋白质分泌表达研究进展

食品级乳酸菌不仅是食品或消化道中传递异源蛋白质的合适的候选菌,在工业发酵中还可用于生产蛋白质。在过去20多年中,人们设计了许多乳酸菌蛋白质表达和标记系统,这些系统已用在乳酸菌工程菌的细胞内或细胞外生产各种细菌、病毒和真核生物来源的蛋白质。在目的蛋白生产和发酵中,分泌表达由于可持续培养和简化纯化步骤并使目的蛋白与其靶位相互作用而优于细胞质表达。目前只有少数研究报道了目的蛋白在乳酸菌细胞内或分泌表达产量的比较,研究表明分泌表达比细胞质表达更优越。     

利用放线菌酮提高黄素单氧化酶FMO_(GS-OX1)亚细胞定位的准确性

研究蛋白质亚细胞定位的常规方法是构建由35S启动子驱动目的基因与绿色荧光蛋白基因(GFP)融合的表达载体,在细胞中瞬时或稳定表达来确定该蛋白质在细胞中的定位。35S启动子的优势是能够获得较强的GFP信号,但同时也可能因为蛋白质合成量过大,导致部分蛋白滞留在运输途径中或出现在其真实定位以外的区域。为了解决这一问题,以模式植物拟南芥中黄素单氧化酶FMOGS-OX1为例,利用蛋白质抑制剂放线菌酮处理过量表达FMOGS-OX1-GFP融合蛋白的烟草叶片。结果表明:未经放线菌酮处理的烟草叶片表皮细胞,细胞质和内质网中均呈现了较强的荧光信号,放线菌酮处理后,内质网上的信号消失,而细胞质则呈现出稳定的信号,因此判断FMOGS-OX1合成后可能是经过内质网运输到细胞质中的。上述结果证明适当的放线菌酮处理,能够避免强启动子驱动报告基因造成的蛋白质合成过量的问题,可有效地提高蛋白质亚细胞定位的准确性。     

无细胞蛋白质合成系统的研究进展

无细胞蛋白质合成系统是一种以外源mRNA或DNA为模板 ,通过在细胞抽提物的酶系中补充底物和能源物质来合成蛋白质的体外系统 .与传统的体内重组表达系统相比 ,体外无细胞合成系统具有多种优点 ,如可表达对细胞有毒害作用或含有非天然氨基酸 (如D 氨基酸 )的特殊蛋白质 ,能够直接以PCR产物作为模板同时平行合成多种蛋白质 ,开展高通量药物筛选和蛋白质组学的研究等 .本文综述了无细胞蛋白质合成系统的发展历史、系统中合成蛋白质所需的能量供应、遗传模板的稳定性和微型无细胞生物反应器等多方面的研究 ,并探讨了无细胞蛋白质合成系统中存在的难点、研究方向和广泛的应用前景     

FABP5基因重组突变体蛋白抗前列腺癌细胞活性分析

为构建脂肪酸结合蛋白5 (FABP5)突变体的原核表达体系,评价突变体蛋白质体外抗前列腺癌细胞的活性.利用定点突变技术,突变FABP5蛋白脂肪酸结合的3个关键位点,并构建原核表达体系,对重组蛋白质进行原核表达、分离纯化.通过细胞毒性、细胞划痕和细胞侵袭试验,评价重组FABP5突变体蛋白质对前列腺癌细胞22RV1和PC3增殖、迁移和侵袭的影响.结果 显示定点突变后的DNA与表达载体pQE32连接并转入大肠杆菌(Escherichia coli) BL21(DE3),经序列测定正确,构建了重组表达工程菌,并诱导表达的重组蛋白经亲和层析纯化获得纯度较高的重组蛋白;三突变体对细胞的增殖、迁移和侵袭抑制作用比3个单突变体和3个双突变体抑制效果明显;单突变体和双突变体组内对细胞的增殖、迁移和侵袭抑制作用差异较小;而野生型重组蛋白质对两株细胞的增殖、迁移和侵袭具有促进作用.本研究从所有突变体中筛选出FABP5的三突变体重组蛋白质对前列腺癌细胞抑制作用较好,为后续开发去势抵抗性前列腺癌(CRPC)蛋白质药物提供参考.     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白。从麻疯树种子中分离得到一种分子量为28.2kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC_(50)为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性。依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5′-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列。该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽。推测的多肽序列与测定的蛋白质N端序列相同,与多种已发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性。将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达。将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力。     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白.从麻疯树种子中分离得到一种分子量为28.2 kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC50为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性.依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5'-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列.该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽.推测的多肽序列与测定的蛋白质N端序列相同,与多种己发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性.将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达.将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力.     

信号肽序列及其在蛋白质表达中的应用

信号肽在蛋白分泌的过程中起重要作用,分泌性蛋白质合成后由信号肽引导其穿过合成所在的细胞到其他组织细胞中。可以利用因特网在线工具和信号序列捕获系统来判定基因序列中是否含有信号肽序列。外源蛋白的表达形式多为细胞内不溶性表达(包涵体),少数为细胞外分泌表达。利用信号肽来引导外源蛋白分泌可避免因包涵体复性带来的困难。研究表明,多种外源基因连接上信号肽后在原核表达系统如大肠杆菌、L型细菌、芽孢杆菌和乳酸杆菌中等都得到了分泌表达;信号肽也广泛应用于真核表达系统如毕赤酵母和昆虫杆状病毒表达系统,以提高蛋白的表达量。     

人造细胞内蛋白质合成的研究进展

无细胞蛋白质合成(cell-free protein synthesis,CFPS)是一种在体外快速合成目标蛋白质的方法,通过构建含有CFPS系统的人造细胞,能够实现蛋白质的高通量表达和功能性膜蛋白的体外重构.本文详细综述了4种CFPS系统(包括大肠杆菌裂解液、兔网织红细胞裂解液、小麦胚芽提取物、酵母提取物)的适用范围和优缺点,总结了基于CFPS系统构建的人造细胞体系内蛋白质合成的研究现状,以及该领域面临的挑战及未来的发展方向.     

Finding one of a kind: advances in single-protein production

An ultimate goal for any protein production system is to express only the protein of interest without producing other cellular proteins. To date, there are only two established methods that will allow the successful expression of only the protein of interest: the cell-free in vitro protein synthesis system and the in vivo single-protein production (SPP) system. Although single-protein production can be achieved in cell-free systems, it is not easy to completely suppress the production of cellular proteins during the production of a protein of interest in a living cell. However, the finding of a unique sequence-specific mRNA interferase in Escherichia coli led to the development of the SPP system by converting living cells into a bioreactor that produces only a single protein of interest without producing any cellular proteins. This technology not only provides a new high expression system for proteins, but also offers a novel avenue for protein structural studies.     

Single protein production (SPP) system in Escherichia coli

Here, we provide a detailed protocol for the single protein production (SPP) system, which is designed to produce only a single protein of interest in living Escherichia coli cells. Induction of MazF, an mRNA interferase that cleaves RNA at ACA nucleotide sequences, results in complete cell growth arrest. However, if mRNA encoding a protein of interest is engineered to be devoid of ACA base triplets and is induced at 15 degrees C using pCold vectors in MazF-expressing cells, only the protein from this mRNA is produced at a yield of 20-30% of total cellular protein; other cellular protein synthesis is almost completely absent. In theory, any protein can be produced by the SPP system. Protein yields are typically unaffected even if the culture is condensed up to 40-fold, reducing the cost of protein production by up to 97.5%. The SPP system has a number of key features important for protein production, including high-yield and prolonged production of isotope-labeled protein at a very high signal-to-noise ratio. The procedure can be completed in 7 d after cloning of an ACA-less target gene into the expression system.     

Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.     

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the addition of an inducer (IPTG [isopropyl-β-d-thiogalactopyranoside]), the mutated MazF [MazF(ΔW)] can still be produced even in the absence of tryptophan in the medium by using a Trp auxotroph, while a target protein having Trp residues cannot be produced. However, at 3 h after the addition of IPTG, the addition of tryptophan to the medium exclusively induces production of the target protein at a high level. A similar SPP system was also constructed with the use of a His-less protein [MazF(ΔH)] and a His auxotroph. Using these dual-induction systems, isotopic enrichments of ¹³C, ¹⁵N, and ²H were highly improved by almost complete suppression of the production of the unlabeled target protein. In both systems, isotopic incorporation reached more than 98% labeling efficiency, significantly reducing the background attributable to the unlabeled target protein.The condensed single-protein production (cSPP) system was developed based on the endoribonuclease activity of an Escherichia coli toxin called MazF, which selectively cleaves cellular mRNAs at the ACA codon sequence (14, 16). Upon induction of MazF, protein synthesis is completely inhibited, and as a result, cell growth is also completely arrested. However, MazF-induced cells are in a quasidormant state, as they are metabolically fully active, producing ATP, amino acids, and nucleotides. Most significantly, in the quasidormant cells, machineries for protein synthesis and mRNA production are also fully functional, and therefore the MazF-induced quasidormant cells are still capable of synthesizing a protein of interest without producing any other cellular proteins, if the mRNA for the protein is engineered to have no ACA sequences (14). This system is thus termed the single-protein production (SPP) system. One of the most remarkable advantages of the SPP system is that the cell culture can be highly condensed without affecting protein yields (7, 12, 13). Using this cSPP system, one can achieve a cost savings of as much as 97.5% by condensing a culture 40-fold. This is particularly valuable when highly expensive isotopes or isotope-labeled compounds, such as amino acids and glucose, are used for the preparation of protein samples for structural study by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, by use of the cSPP system, amino acid analogues or D₂O, which is toxic in conventional protein production systems, reducing protein yields, is not toxic, hardly affecting the final protein yields. However, one drawback of the current cSPP system is the use of IPTG (isopropyl-β-d-thiogalactopyranoside) as an inducer for both MazF and a target protein, such that the target protein is also produced at the same time as MazF. Since isotopes or isotope-labeled compounds are added 2 to 3 h after the addition of IPTG to avoid their incorporation into cellular proteins, non-isotope-labeled target protein is also produced during this preincubation period, resulting in a higher background of unlabeled target protein, which may be as high as 20% of the final yield of the target protein produced (10).The combination of both tetracycline- and IPTG-inducible systems was employed to separate the inductions of target protein and MazF, respectively (10). The main disadvantage of this system is that the expression level of target protein critically depends upon tetracycline concentration. The amounts of tetracycline being added to the cells for induction of target protein significantly affect the level of target protein synthesis, especially in the condensed SPP system. Therefore, a highly precise and accurate optimization of the tetracycline level is required for consistency in the expression of target proteins. To circumvent this problem, we developed a novel dual-induction system using amino acid auxotrophs. It has been shown previously that E. coli cells from a histidine (His) auxotroph can still produce a protein containing no His residues in the absence of histidine in the medium without producing any other cellular proteins (5). Therefore, it is assumed that even if both MazF and a target protein containing His residues or tryptophan (Trp) residues are coinduced by IPTG, only His-less MazF or Trp-less MazF would be produced in the absence of histidine or tryptophan by using a His auxotroph or a Trp auxotroph, respectively. In this fashion, the target protein may be induced by the addition of histidine or tryptophan in the medium a few hours after His-less or Trp-less MazF induction so that background production of the target protein may be avoided. Furthermore, this new SPP system could also be useful in studies involving specific replacement of amino acids with their analogues.In the present study, we employed two amino acid auxotrophs, of Trp and His, to construct the dual-induction SPP system. For this purpose, both Trp-less proteins and His-less MazF proteins [MazF(ΔW) and MazF(ΔH), respectively] were generated to create the new SPP system. Using this new system, we tested a number of proteins, such as (i) E. coli EnvZB, which is the ATP-binding domain of the histidine kinase EnvZ (161 residues) (15), (ii) E. coli CspA, which is the major cold shock protein (70 residues) (3), (iii) E. coli YaiZ, which is a plasma membrane protein (80 residues) (7), (iv) the antiapoptotic adenoviral protein E1B19K150 (150 residues) (2), (v) human granulocyte colony-stimulating factor (GCSF; 175 residues) (4), and (vi) human calmodulin (CaM), which is a calcium binding protein (148 residues) (6). It was found that isotope incorporation into these proteins was very tightly regulated so that the background due to the unlabeled target protein was significantly reduced. We also developed a pCold(W) system in which a Trp tag can be added to the N-terminal part of a protein so that the dual-induction expression can still be applied for Trp-less proteins.     

Partner-aware prediction of interacting residues in protein-protein complexes from sequence data

Computational prediction of residues that participate in protein-protein interactions is a difficult task, and state of the art methods have shown only limited success in this arena. One possible problem with these methods is that they try to predict interacting residues without incorporating information about the partner protein, although it is unclear how much partner information could enhance prediction performance. To address this issue, the two following comparisons are of crucial significance: (a) comparison between the predictability of inter-protein residue pairs, i.e., predicting exactly which residue pairs interact with each other given two protein sequences; this can be achieved by either combining conventional single-protein predictions or making predictions using a new model trained directly on the residue pairs, and the performance of these two approaches may be compared: (b) comparison between the predictability of the interacting residues in a single protein (irrespective of the partner residue or protein) from conventional methods and predictions converted from the pair-wise trained model. Using these two streams of training and validation procedures and employing similar two-stage neural networks, we showed that the models trained on pair-wise contacts outperformed the partner-unaware models in predicting both interacting pairs and interacting single-protein residues. Prediction performance decreased with the size of the conformational change upon complex formation; this trend is similar to docking, even though no structural information was used in our prediction. An example application that predicts two partner-specific interfaces of a protein was shown to be effective, highlighting the potential of the proposed approach. Finally, a preliminary attempt was made to score docking decoy poses using prediction of interacting residue pairs; this analysis produced an encouraging result.     

Triathlon for energy functions: Who is the winner for design of protein–protein interactions?

Computational prediction of stabilizing mutations into monomeric proteins has become an almost ordinary task. Yet, computational stabilization of protein–protein complexes remains a challenge. Design of protein–protein interactions (PPIs) is impeded by the absence of an energy function that could reliably reproduce all favorable interactions between the binding partners. In this work, we present three energy functions: one function that was trained on monomeric proteins, while the other two were optimized by different techniques to predict side-chain conformations in a dataset of PPIs. The performances of these energy functions are evaluated in three different tasks related to design of PPIs: predicting side-chain conformations in PPIs, recovering native binding-interface sequences, and predicting changes in free energy of binding due to mutations. Our findings show that both functions optimized on side-chain repacking in PPIs are more suitable for PPI design compared to the function trained on monomeric proteins. Yet, no function performs best at all three tasks. Comparison of the three energy functions and their performances revealed that (1) burial of polar atoms should not be penalized significantly in PPI design as in single-protein design and (2) contribution of electrostatic interactions should be increased several-fold when switching from single-protein to PPI design. In addition, the use of a softer van der Waals potential is beneficial in cases when backbone flexibility is important. All things considered, we define an energy function that captures most of the nuances of the binding energetics and hence, should be used in future for design of PPIs.     

A novel 76-mer peptide mimic with the synergism of superoxide dismutase and glutathione peroxidase

The balance of oxidation and reduction in the body requires the synergistic effect of various antioxidant enzymes. Therefore, the construction of enzyme mimics with multiple antioxidant activities is important and beneficial for further research on the synergistic effects of antioxidant enzymes and their mechanism of action. To explore the synergistic effect of superoxide dismutase (SOD) and glutathione peroxidase (GPx), a 76-mer selenium-containing peptide (Se-76P) mimic containing the active SOD and GPx centers was designed. Moreover, a cell-penetrating peptide was introduced into Se-76P by structure modeling, and then, Se-76P was expressed by a single-protein production combined with the cysteine auxotrophic double-expression system of Escherichia coli. The results suggest that Se-76P exhibits SOD and GPx activities, following the GPx activity of 109 U/mg protein and the SOD activity of 1218 U/mg protein. The labeled Se-76P with FITC fluorescence was verified to enter the L02 cells successfully; it improved the antioxidant activity in cells and promoted the consumption of glucose and synthesis of glycogen. The injection of Se-76P subcutaneously decreased the levels of blood glucose and malondialdehyde of lipid peroxidation produced in mice, indicating that Se-76P had antioxidative properties and a certain regulatory role of glucose metabolism. The data analysis provides further clarification that Se-76P can regulate insulin signal transduction to play an insulin-like role, which not only has a greater significance for further elucidating the catalytic mechanism of the enzyme and their synergistic effects on each other but also has enormous medicinal potential.     

激光拉曼光谱在蛋白质构象研究中的应用和进展

激光拉曼光谱法被公认为是研究生物大分子的结构、动力学和功能的有效方法。近年来拉曼光谱在蛋白质构象研究中的最新进展,涉及到拉曼光谱在非折叠蛋白质、蛋白质装配的特征描述,拉曼晶体学在实时监控蛋白质单晶中化学变化等方面的应用。另外,介绍了蛋白质拉曼光谱分析在生物技术中的应用现状。并对拉曼光谱技术在蛋白质等生物大分子领域中的研究前景做了进一步的展望。     

Protein salting-out: phase equilibria in two-protein systems

The phase behavior of two aqueous binary protein mixtures, lysozyme-chymotrypsin and lysozyme-ovalbumin, was determined in ammonium sulfate solutions. Protein concentrations were determined in both phases as a function of pH and ionic strength. For lysozyme-chymotrypsin mixtures, the observed phase behavior was similar to that for each individual protein; the presence of the second protein had little influence. The phase behavior of lysozyme-ovalbumin mixtures, however, was different from that of the respective single-protein systems. Lysozyme and ovalbumin are found together in egg whites; their association is both pH and ionic-strength dependent. The association of proteins is a key determinant of protein solubility in salt solutions. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 567-574, 1997.