无细胞蛋白质合成系统的研究进展及应用前景

无细胞蛋白质合成系统是现代迅速发展的蛋白质表达系统,以细胞抽提物中酶和蛋白质因子等作为基本反应体系,添加外源模板、底物以及能源物质等维持体系运作,最终在体外合成目标蛋白质。无细胞蛋白质合成系统突破了细胞的生理限制,能够灵活地进行蛋白质合成,极大地提高了蛋白质的产量,不仅可以作为研究转录和翻译的研究工具,而且可以实现蛋白质的高通量表达,在诸多领域带来了突破性的进展。现主要针对无细胞蛋白质合成系统的能量供给、蛋白质的稳定性和折叠修饰以及应用进行综述,以期推动对该技术的理解和应用。     

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.     

无细胞蛋白质合成的研究进展

无细胞合成生物学作为新兴的多学科交叉手段,已很好地应用于蛋白质工程领域。因为无细胞生物合成系统无需完整的活细胞,在体外就可激活转录和翻译机器,因此,可减少对细胞的依赖性,从而增加工程的自由度。无细胞系统的这些优点使它已经成为强大的蛋白质工程平台,用于膜蛋白和医药蛋白的合成、非天然氨基酸的嵌入以及高通量分析。无细胞蛋白质合成系统不仅在基础科学研究而且在工业化生产中拥有广阔的应用前景。本文中,笔者系统综述了无细胞蛋白质合成系统的研究及应用,并展望了该系统的研究前景。     

无细胞合成生物学：革新生物医药产业的新策略

无细胞合成生物系统,能够在体外完成生命转录翻译过程,因体系灵活开放、便于控制、表达周期短、高耐受性等特点,可表达细胞系统难以表达的蛋白质。随着无细胞生物传感和体系冻干技术的不断发展,其在医药健康领域的应用不断拓展。本文综述了无细胞合成生物学在按需生物医药合成和便携式医疗检测等医药健康领域的研究进展,该体系的进一步发展有潜力实现更复杂后修饰蛋白质药物的合成、可丰富无细胞生物传感器类型并提高其灵敏性。无细胞合成生物学作为新兴工程策略,未来必将更好地应用于高通量医药蛋白质筛选、新型病原体的检测等医药健康领域。     

无细胞蛋白质合成系统

无细胞蛋白质合成系统相比传统的细胞表达系统有许多优点,包括表达周期短、反应条件容易控制等。该文介绍了无细胞蛋白质合成系统的技术进展及表达控制,并综述了这一系统的应用发展。     

体外翻译系统在分子生物学研究中的应用

体外翻译系统又称无细胞蛋白质合成系统,是分子生物学中一种常规的表达系统,该系统可用于蛋白质快速分析鉴定,基因转录和翻译的调控机理的研究以及分子间的相互作用的研究,如蛋白质和蛋白质的相互作用,蛋白质与DNA的相互作用,蛋白质与RNA的相互作用等。     

无细胞体系非天然蛋白质合成研究进展

无细胞非天然蛋白质合成作为蛋白质研究的新兴手段,已成功用于表征蛋白质分子间、蛋白质与核酸分子间相互作用等基础科学研究及医药蛋白、蛋白质材料等工业生产领域。无细胞非天然蛋白质合成系统不需维持细胞的生长,无细胞膜阻碍,可依据研究目的添加基因元件或化学物质从而增强工程设计和过程调控的自由性;也可赋予蛋白质新的特性、结构及功能,如可实现蛋白翻译后修饰、反应手柄引入、生物物理探针及多聚蛋白质合成等。文中系统地综述了目前应用于无细胞蛋白质合成系统中的非天然氨基酸嵌入方法,包括全局抑制及基于正交翻译体系的终止密码子抑制、移码抑制、有义密码子再分配和非天然碱基等方法的研究进展,及非天然氨基酸在蛋白质修饰、生物物理探针、酶工程、蛋白质材料以及医药蛋白质生产等领域的应用进展,并分析了该体系的发展前景及广泛工业化应用的机遇与挑战。     

无细胞蛋白合成系统的研究进展

无细胞蛋白合成系统能够以DNA或RNA为模板直接在体外合成蛋白,通过种种技术改进,在某些个例中,蛋白表达量已接近体内表达水平,并且建立了翻译后修饰和蛋白纯化的方法,进一步提高无细胞系统的合成能力,会使它比重组细胞的工业生产更具竞争力。     

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.     

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。     

Regulation of synthesis of a brain-specific protein in monolayer cultures of clonal rat glial cells

C6 cells were grown in monolayer culture under conditions permitting continued exponential cell division after attainment of a density at which extensive intercellular contacts were formed. An increase in the relative synthesis of S100 protein coincided with the time of formation of extensive intercellular contacts and preceded the onset of the stationary phase of growth by three generations. These observations suggested that the induction of S100 protein synthesis was mediated by cell contact and not by an arrest of cellular growth. The mechanism of this induction was first studied in a homologous non-initiating cell-free protein-synthesizing system from C6 cells, using fixed amounts of free amino acids or fully charged rat liver aminoacyl-tRNA as a source of precursors for protein synthesis. Real synthesis of total soluble proteins decreased as the cells progressed from logarithmic to stationary growth while synthesis of S100 protein increased during this period. The capacity of poly(A)+ RNA from logarithmic and stationary cultures to direct the synthesis of S100 protein was estimated in a cell-free protein-synthesizing system derived from wheat embryos. Increased synthesis of S100 protein in stationary cultures was directly correlated with an increase in translatable S100 protein mRNA.     

Development of a rapid and productive cell-free protein synthesis system

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast toin vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately 650 μg/mL of protein was produced after 2 h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.     

Dissection of individual functions of the Sendai virus phosphoprotein in transcription.

The Sendai virus P protein is an essential component of the viral RNA polymerase (P-L complex) required for RNA synthesis. To identify amino acids important for P-L binding, site-directed mutagenesis of the P gene changed 17 charged amino acids, singly or in groups, and two serines to alanine within the L binding domain from amino acids 408 to 479. Each of the 10 mutants was wild type for P-L and P-P protein interactions and for binding of the P-L complex to the nucleocapsid template, yet six showed a significant inhibition of in vitro mRNA and leader RNA synthesis. To determine if binding was instead hydrophobic in nature, five conserved hydrophobic amino acids in this region were also mutated. Each of these P mutants also retained the ability to bind to L, to itself, and to the template, but two gave a severe decrease in mRNA and leader RNA synthesis. Since all of the mutants still bound L, the data suggest that L binding occurs on a surface of P with a complex tertiary structure. Wild-type biological activity could be restored for defective polymerase complexes containing two P mutants by the addition of wild-type P protein alone, while the activity of two others could not be rescued. Gradient sedimentation analyses showed that rescue was not due to exchange of the wild-type and mutant P proteins within the P-L complex. Mutants which gave a defective RNA synthesis phenotype and could not be rescued by P establish an as-yet-unknown role for P within the polymerase complex, while the mutants which could be rescued define regions required for a P protein function independent of polymerase function.     

Evidence for the presence of mRNA in the post-ribosomal cytoplasm of sheep lymphocytes.

Several fractions of RNA prepared from the post-ribosomal cytosol of sheep lymphoid cells were found to include messenger-like RNA as defined by the following criteria: a, template activity, i.e. the ability to promote the incorporation of radioactive amino acids into protein in cell-free protein-synthesising systems derived from wheat embryos or ascites tumour cells; b, a low magnesium optimum (1-2.5 mM) for template activity which is characteristic of many natural mRNAs; c, sensitivity of the template response to aurintricarboxylic acid, a specific inhibitor of the initiation of protein synthesis. The lymphoid post-ribosomal RNA fractions, however, were translated less efficiently than were rabbit reticulocyte globin mRNA or tobacco mosaic viral (TMV) RNA; no explanation for this relatively poor template activity was found. The major fraction of messenger-like RNA had an average sedimentation coefficient of 12 S; this fraction directed the translation of several discrete polypeptides in the molecular weight range 10 000-25 000. On average the products of 12 S RNA-directed protein synthesis appeared lysine rich compared with TMV RNA-directed products. It is suggested that the apparent pool of uncommitted mRNA in resting lymphocytes may be utilised during the early stages of lymphocyte activation, and that the mRNAs could be stored in forms similar to those evident in other dormant tissues.     

Identification of the critical region in replication factor C from Pyrococcus furiosus for the stable complex formation with proliferating cell nuclear antigen and DNA

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis. The function of RFC is to load PCNA, a processivity factor of replicative DNA polymerases, onto primed DNA templates. The central hole of the PCNA homo-trimeric ring encircles doublestranded DNA, so that DNA polymerases can operate for DNA synthesis with PCNA along a DNA template. The Pyrococcus furiosus RFC (PfuRFC) consists of a small subunit (RFCS, 37kDa) and a large subunit (RFCL, 55kDa), which show significant sequence identity to the eukaryotic homologs. The C-terminal region of RFCL has an acidic cluster of about 30 amino acids, which consists mainly of glutamic acid residues, and a following basic cluster of 10 amino acids, which consists mainly of lysine residues. These clusters of charged amino acids, which precede the C-terminal consensus sequence, PIP (PCNA interacting protein)-box, are conserved in several archaeal RFCLs. The series of mutant PfuRFC containing the C-terminal deletions in RFCL were constructed. The mutational analyses showed that the charged cluster is not essential for loading of PCNA onto DNA. However, the region containing the basic cluster is important for the stable ternary (RFC-PCNA-DNA) complex formation.     

The natural non-protein amino acid N-β-methylamino-l-alanine (BMAA) is incorporated into protein during synthesis

N-β-methylamino-l-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC–MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.     

Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing.

The complete sequence of the mouse immunoglobulin kappa light chain MOPC 21 messenger RNA has been determined using a chain termination method and chemically synthesised deoxyoligonucleotides to initiate the synthesis of a DNA molecule complementary to the mRNA template. Five such oligonucleotide primers have been used for the sequence analysis of this messenger RNA. The approach is excellent for comparative studies of mouse k-chain mRNAs because they can be made on impure mRNA preparations. The MOPC 21 light chain mRNA is 943 nucleotides in length excluding the poly(A) region. An unexpected finding was that there are only three bases in the 5' non-coding region and its significance in terms of ribosome binding is discussed; 87 code for the precursor or leader sequence of the protein, 642 for the mature protein and 211 for the 3' non-coding region. The codons for the precursor region allows the previously undetermined amino acid sequence to be predicted. In common with other precursor regions a high proportion of the predicted amino acids are hydrophobic.     

Cell-free protein synthesis: the state of the art

Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.