Site-specific protein modification: advances and applications

Although chemical methods to modify proteins in a sequence-specific manner have yet to be developed, site-specific post-translational modification of proteins has recently emerged as a major focus in biological chemistry. Post-translational modification with functionalized substrate analogues opens up several unique avenues to induce selective reactivity into proteins in a sequence-specific manner, and can be applied to protein identification and manipulation in both in vitro and in vivo contexts. Further in vivo applications of this method will enable the imaging of cellular processes, avoiding nonspecific labeling and probe scattering, major complications observed in nonenzymatic methods. Additionally, new tools for in vitro protein modification have been developed that offer more versatile ways to study protein structure and function.     

Chlamydomonas reinhardtii

Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

综述与专论：遗传密码子扩展技术在蛋白质及多肽类药物中的应用

通过遗传密码子扩展技术位点特异性插入非天然氨基酸(noncanonical amino acids,ncAAs)可在原子水平上对蛋白质的结构与功能进行操控。目前该技术能够向包括高等动植物在内的各种生命体中插入200多种ncAAs,已被广泛应用于生物医药领域。凭借能够在蛋白质中定点引入可控生物正交化学官能团的独特优势,该技术不仅可以用于蛋白质及多肽药物的研发,提高蛋白质及多肽药物的质量与疗效,而且可以为一些人类重大疾病的预防和治疗提供开创性解决方案。本文将重点关注遗传密码子扩展技术的前沿进展及其在各类抗体、细胞因子以及抗菌肽等蛋白质及多肽类药物中的应用,同时也对其衍生的新型生物治疗手段进行简单阐述。     

内含肽介导的蛋白连接技术及其应用

内含肽介导的蛋白质剪接是一种自发的翻译后事件,内含肽可介导其自身从前体蛋白上切除,同时将其两侧的外显肽连接起来.在过去10多年中,基于蛋白质剪接原理发展出的蛋白连接技术被广泛的用于蛋白质工程的研究中.这些技术打破了化学合成方法中对目标物大小的限制,有助于化学和生物学的研究.针对近年由蛋白质连接演化出来的新技术及其应用做简要的阐述.     

Intein-mediated ligation and cyclization of expressed proteins

Protein splicing is a posttranslational processing event that releases an internal protein sequence from a protein precursor. During the splicing process the internal protein sequence, termed an intein, embedded in the protein precursor self-catalyzes its excision and the ligation of the flanking protein regions, termed exteins. The dissection of the splicing pathway, which involves the precise cleavage and formation of peptide bonds, and the identification of key catalytic residues at the splice junctions have led to the modulation of the protein splicing process as a protein engineering tool. Novel strategies have been developed to use intein-catalyzed reactions for the production and manipulation of proteins and peptides. These new approaches have broken down the size limitation barrier of chemical synthetic methods and are less technically demanding. The purpose of this article is to describe how to use self-splicing inteins in protein semisynthesis and backbone cyclization. The first two sections of the article provide a brief review of the distinct chemical steps that underlie protein splicing and intein enabled technology.     

Genetic screens and directed evolution for protein solubility

Overexpressed proteins are often insoluble, and can be recalcitrant to conventional solubilization techniques such as refolding. Directed evolution methods, in which protein diversity libraries are screened for soluble variants, offer an alternative route to obtaining soluble proteins. Recently, several new protein solubility screens have been developed that do not require structural or functional information about the target protein. Soluble protein can be detected in vivo and in vitro by fusion reporter tags. Protein misfolding can be measured in vivo using the bacterial response to protein misfolding. Finally, soluble protein can be monitored by immunological detection. Efficient, well-established strategies for generating and recombining genetic diversity, driven by new screening and selection methods, can furnish correctly folded, soluble protein.     

TAT-mediated protein transduction into mammalian cells

Manipulation of mammalian cells has been achieved by the transfection of expression vectors, microinjection, or diffusion of peptidyl mimetics. While these approaches have been somewhat successful, the classic manipulation methods are not easily regulated and can be laborious. One approach to circumvent these problems is the use of HIV TAT-mediated protein transduction. Although this technology was originally described in 1988, few improvements were reported in the subsequent 10 years. In the last few years, significant steps have been taken to advance this technology into a broadly applicable method that allows for the rapid introduction of full-length proteins into primary and transformed cells. The technology requires the synthesis of a fusion protein, linking the TAT transduction domain to the molecule of interest using a bacterial expression vector, followed by the purification of this fusion protein under either soluble or denaturing conditions. The purified fusion protein can be directly added to mammalian cell culture or injected in vivo into mice. Protein transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 5 min, with nearly equal intracellular concentrations between all cells in the transduced population. Full-length TAT fusion proteins have been used to address a number of biological questions, relating to cell cycle progression, apoptosis, and cellular architecture. Described here are the fundamental requirements for the creation, isolation, and utilization of TAT-fusion proteins to affect mammalian cells. A detailed protocol for production and transduction of TAT-Cdc42 into primary cells is given to illustrate the technique.     

Ribosome display: cell-free protein display technology.

Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. It uses the principle of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes), through the formation of stable protein-ribosome-mRNA (PRM) complexes. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA, which is then converted and amplified as DNA for further manipulation, including repeated cycles or protein expression. Ribosome display has a number of advantages over cell-based systems such as phage display; in particular, it can display very large libraries without the restriction of bacterial transformation. It is also suitable for generating toxic, proteolytically sensitive and unstable proteins, and allows the incorporation of modified amino acids at defined positions. In combination with polymerase chain reaction (PCR)-based methods, mutations can be introduced efficiently into the selected DNA pool in subsequent cycles, leading to continuous DNA diversification and protein selection (in vitro protein evolution). Both prokaryotic and eukaryotic ribosome display systems have been developed and each has its own distinctive features. In this paper, ribosome display systems and their application in selection and evolution of proteins are reviewed.     

An incremental approach to automated protein localisation

Background  

The subcellular localisation of proteins in intact living cells is an important means for gaining information about protein functions. Even dynamic processes can be captured, which can barely be predicted based on amino acid sequences. Besides increasing our knowledge about intracellular processes, this information facilitates the development of innovative therapies and new diagnostic methods. In order to perform such a localisation, the proteins under analysis are usually fused with a fluorescent protein. So, they can be observed by means of a fluorescence microscope and analysed. In recent years, several automated methods have been proposed for performing such analyses. Here, two different types of approaches can be distinguished: techniques which enable the recognition of a fixed set of protein locations and methods that identify new ones. To our knowledge, a combination of both approaches – i.e. a technique, which enables supervised learning using a known set of protein locations and is able to identify and incorporate new protein locations afterwards – has not been presented yet. Furthermore, associated problems, e.g. the recognition of cells to be analysed, have usually been neglected.     

Challenges in mass spectrometry-based proteomics

During the last decade, protein analysis and proteomics have been established as new tools for understanding various biological problems. As the identification of proteins after classical separation techniques, such as two-dimensional gel electrophoresis, have become standard methods, new challenges arise in the field of proteomics. The development of "functional proteomics" combines functional characterization, like regulation, localization and modification, with the identification of proteins for deeper insight into cellular functions. Therefore, different mass spectrometric techniques for the analysis of post-translational modifications, such as phosphorylation and glycosylation, have been established as well as isolation and separation methods for the analysis of highly complex samples, e.g. protein complexes or cell organelles. Furthermore, quantification of protein levels within cells is becoming a focus of interest as mass spectrometric methods for relative or even absolute quantification have currently not been available. Protein or genome databases have been an essential part of protein identification up to now. Thus, de novo sequencing offers new possibilities in protein analytical studies of organisms not yet completely sequenced. The intention of this review is to provide a short overview about the current capabilities of protein analysis when addressing various biological problems.     

蚜虫唾液蛋白研究进展

蚜虫属于半翅目蚜科,多为重要的农业害虫,通过刺吸式口器吸食植物汁液,传播病毒,其爆发常常造成重大经济损失。在漫长的协同进化历程中,植物建立了高效的防御系统以应对蚜虫威胁。为了克服植物的防御反应,蚜虫也发展了相应的反制手段,其中蚜虫在取食过程中分泌的唾液蛋白能调控植物防御反应,降解植物次生物质,从而在蚜虫与植物互作中发挥着至关重要的作用。本文综述了蚜虫唾液蛋白的组分鉴定方法和相关蛋白的功能,并对唾液蛋白在蚜虫防治的应用和今后的研究方向进行了展望。常见的蚜虫唾液蛋白组分的鉴定和分析方法包括唾液蛋白的酶活性分析、唾液蛋白组学分析、唾液腺转录组学和蛋白组学分析等。但这些方法各有利弊,仅采取一种分析方法不能客观全面地反映蚜虫唾液蛋白分泌谱,多种技术手段联合分析方可提供更为逼真详实的信息。蚜虫唾液蛋白种类繁多,可分为解毒酶、保护酶、水解酶、结合功能蛋白以及分类未知的效应蛋白等。蚜虫唾液蛋白功能多样,能参与唾液鞘的形成,诱导植物防御反应,促进蚜虫取食,提高蚜虫繁殖力等。通过RNAi干扰唾液蛋白编码基因会显著改变蚜虫取食行为,并降低蚜虫存活率、产蚜量和适合度。因此,唾液蛋白是防控蚜虫的理想靶标。目前,采用寄主诱导的基因沉默(host-induced gene silencing, HIGS)技术已培育了数种靶向唾液蛋白基因的高效抗蚜作物品系,展示出了良好的应用前景。从目前研究来看,各种蚜虫唾液蛋白谱急需采用多组学手段联合分析的方法来进行完整解析。各种唾液蛋白的具体功能方面的研究还严重缺乏,需从蚜虫、植物、两者之间的互作等多维度探究唾液蛋白的作用及相关的分子机制,为发展基于蚜虫唾液蛋白调控的蚜虫防治新策略打下基础。     

Asymmetric proteome equalization of the skeletal muscle proteome using a combinatorial hexapeptide library

Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method.     

Dunaliella as an attractive candidate for molecular farming

Pharmaceutical recombinant proteins are widely used in human healthcare. At present, several protein expression systems are available to generate therapeutic proteins. These conventional systems have distinct advantages and disadvantages in protein yielding; in terms of ease of manipulation, the time required from gene transformation to protein purification, cost of production and scaling-up capitalization, proper folding and stability of active proteins. Depending on the research goal and priorities, a special system may be selected for protein expression. However, considering the limited variety of organisms currently used and their usage restrictions, there are still much more pharmaceutical proteins waiting to be economically and efficiently produced. Distinguished biological and technical features of microalgae Dunaliella such as inexpensive medium requirement, fast growth rate, the ease of manipulation, easy scaling up procedure, facility of milking in bioreactors and the ability of post-translational modifications make this microorganism an attractive candidate for molecular farming.     

Protein redesign.

It has been demonstrated that, for a number of proteins, it is possible to dramatically alter the connectivities between elements of secondary structure. Remarkably large loop insertions are tolerated and many redesigns have generated proteins that successfully fold to stable, active structures. Some redesigns have been entirely the choice of the investigators, whereas others have incorporated a randomization and selection step to identify optimal sequences. These studies have provided basic guidelines for the rational manipulation of protein structure and stability, they have allowed the dissection of folding pathways and they have generated proteins with the potential for practical therapeutic applications.     

Molecular farming on rescue of pharma industry for next generations

Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification methods. DNA technology and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant molecular pharming and outline some important challenges that hamper these ambitious biotechnological developments.     

Structure-guided engineering of TGF-βs for the development of novel inhibitors and probing mechanism

The increasing availability of detailed structural information on many biological systems provides an avenue for manipulation of these structures, either for probing mechanism or for developing novel therapeutic agents for treating disease. This has been accompanied by the advent of several powerful new methods, such as the ability to incorporate non-natural amino acids or perform fragment screening, increasing the capacity to leverage this new structural information to aid in these pursuits. The abundance of structural information also provides new opportunities for protein engineering, which may become more and more relevant as treatment of diseases using gene therapy approaches become increasingly common. This is illustrated by example with the TGF-β family of proteins, for which there is ample structural information, yet no approved inhibitors for treating diseases, such as cancer and fibrosis that are promoted by excessive TGF-β signaling. The results presented demonstrate that through several relatively simple modifications, primarily involving the removal of an α-helix and replacement of it with a flexible loop, it is possible to alter TGF-βs from being potent signaling proteins into inhibitors of TGF-β signaling. The engineered TGF-βs have improved specificity relative to kinase inhibitors and a much smaller size compared to monoclonal antibodies, and thus may prove successful as either as an injected therapeutic or as a gene therapy-based therapeutic, where other classes of inhibitors have failed.     

蛋白质间相互作用技术的研究近况

蛋白质间相互作用技术的研究近况黄翠芬叶棋浓（军事医学科学院生物工程研究所,北京１００８５０关键词：蛋白质,相互作用,技术ＲｅｃｅｎｔＡｄｖａｎｃｅｓｉｎｔｈｅＴｅｃｈｎｉｑｕｅｓｏｆＰｒｏｔｅｉｎ┐ＰｒｏｔｅｉｎＩｎｔｅｒａｃｔｉｏｎｓＨｕａｎｇＣｕ...