Functional properties of a recombinant bacterial DING protein: comparison with a homologous human protein

DING proteins are highly-conserved proteins with poorly-defined cell-signalling roles in mammals. Conserved homologues are also commonplace in plants, though not as yet functionally characterized. Poor availability of the proteins, and a lack of genetic structure, hamper progress in elucidating the roles of these eukaryotic DING proteins, but highly-homologous hypothetical DING proteins have recently been identified in Pseudomonas genomes. We have cloned and expressed a DING protein from P. fluorescens SWB25 in Escherichia coli. The recombinant protein, and its natural human homologue, act as phosphate-binding proteins, as predicted by structural homologies with other bacterial proteins. The recombinant protein also displays other functional similarities with mammalian DING proteins, in that, like the human version, it acts as a mitogen for cultured human cells, and can bind cotinine, known to be a binding ligand for a rat neuronal DING protein.     

Potential of plants to produce recombinant protein products

Plants have great potential as photosynthetic factories to produce pharmaceutically important and commercially valuable biomedicines and industrial proteins at low cost. The U.S. Food and Drug Administration (U.S. FDA) has approved the drug Elelyso (taliglucerase alfa) produced by carrot cells for treatment of type 1 Gaucher’s disease in 2012. The commercial potential of biomedicines produced by molecular farming has dramatically improved due to the success of an experimental drug called ZMapp, which has immunological activity in Ebola patients. A cocktail of three monoclonal antibodies was produced in tobacco (Nicotiana benthamiana) plants (Chen and Davis 2016). At present, very few drugs made by this technology have been approved by worldwide authorities such as the U.S. FDA. However, plants have been proposed as a novel paradigm for commercial production of proteins over the next decade. In recent years, leading researchers on molecular farming have given more priority to the area of animal-free therapeutic proteins such as parenteral and oral vaccines. Although plant-based platforms have considerable advantages over traditional systems such as bacterial and animal systems, there are several obstacles to commercial-scale production, especially with regards to improving the quality and quantity of plant-produced biologics and industrial materials. One of the biggest barriers to commercialization of this technology is the intense scrutiny of these new plant varieties by regulatory agencies and the public as well as the high costs associated with their regulatory approval.     

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.     

Rapid purification of a recombinant protein using tandem radial flow ion-exchange column chromatography.

Tandem radial flow anion- and cation-exchange columns were used to partially purify and concentrate a dilute recombinant protein that had been refolded in vitro after production as insoluble inclusion bodies in E. coli. The refolded sample was first passed through a Q-Sepharose Fast Flow column in order to remove the majority of E. coli contaminating proteins and endotoxins, then purified on an S-Sepharose Fast Flow column connected to the outlet of the Q-Sepharose column. This tandem arrangement enabled the rapid processing of multiple preparations of refolded material during production method development.     

Rapid detection of protein tyrosine kinase activity in recombinant yeast expressing a universal substrate

Yeast two-hybrid systems are powerful proteomics tools for the discovery of protein-protein interactions. However, these systems are typically unable to detect interactions dependent on post-translational modifications such as tyrosine phosphorylation. We report a novel yeast tribrid system that expresses a potentially universal protein tyrosine kinase (PTK) substrate to detect diverse PTKs. Validation with the oncogenic kinases v-Abl and v-Src, which exhibit divergent substrate specificities, demonstrated significant potential for cloning PTKs en masse from cDNA libraries.     

Rapid evaluation and optimization of recombinant protein production using GFP tagging

The isolation of recombinant proteins from bacterial or eukaryotic systems often requires a laborious optimization of expression and purification conditions. To greatly facilitate this procedure we included the green fluorescent protein (GFP) in bacterial expression vectors. This approach allowed us to sensitively detect the GFP hybrid proteins already in intact bacterial cells using a fluorescence microscope. To rapidly analyze a variety of conditions essential for protein expression, the GFP signal, indicative of expression levels, was directly quantitated in live bacterial suspensions using a fluorescence plate reader. Thus, GFP tagging not only allows one to directly monitor protein expression in general but also appears to increase protein stability or solubility.     

Habitat monitoring in Europe: a description of current practices

Monitoring of biodiversity at the level of habitats is becoming increasingly common. Here we describe current practices in habitat monitoring based on 150 schemes in Europe. Most schemes were initiated after 1990 in response to EU nature directives or habitat management/restoration actions, with funding mostly from European or national sources. Schemes usually monitor both the spatial distribution and the quality of the habitats, and they frequently collect data on environmental parameters and potential causes of changes. Many schemes are local or regional rather than national or international in scope, and sampling effort varies greatly across spatial and temporal scales. Experimental design is used in half of the schemes, however, data are rarely analysed by advanced statistics. Most schemes require two months or less per year in manpower and are typically run by professionals rather than by volunteers. Estimated salaries plus equipment costs average 650,000 Euro per year per scheme, and add up to 80 million Euros annually. Costs are particularly high for schemes based on European or international law and for schemes funded by European or national sources. Costs are also high in schemes in which sampling sites are selected subjectively rather than based on sampling theory, and in schemes that do not use field mapping or remote sensing to document spatial variation in habitats. Our survey demonstrates promising developments in European habitat monitoring but also underlines the need for better spatial coverage, documentation of spatial variaton, improved sampling design and advanced data analysis. Such improvements are essential if we are to judge progress towards the 2010 biodiversity targets.     

Real-time remote monitoring of water quality: a review of current applications, and advancements in sensor, telemetry, and computing technologies

Recent advances in communication and sensor technology have catalyzed progress in remote monitoring capabilities for water quality. As a result, the ability to characterize dynamic hydrologic properties at adequate temporal and spatial scales has greatly improved. These advances have led to improved statistical and mechanistic modeling in monitoring of water quality trends at local, watershed and regional scales for freshwater, estuarine and marine ecosystems. In addition, they have greatly enhanced rapid (e.g., real-time) detection of hydrologic variability, recognized as a critical need for early warning systems and rapid response to harmful algal bloom events. Here, we present some of the landmark developments and technological achievements that led to the advent of real-time remote monitors for hydrologic properties. We conclude that increased use and continuing advancements of real-time remote monitoring (RTRM) and sensing technologies will become a progressively more important tool for evaluating water quality. Recent engineering and deployment of RTRM technologies by federal and state regulatory agencies, industries, and academic laboratories is now permitting rapid detection of, and responses to, environmental threats imposed by increased nutrient loadings, development of hypoxic and anoxic areas, toxicants, and harmful algal bloom outbreaks leading to fish kill events and potential human health impacts.     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.Key words: biosimilar mAb, innovator mAb, molecular similarity, sequence variants, posttranslational modifications, N-linked glycosylation, chemical degradations, micro-heterogeneities, characterization, intact protein mass measurement, peptide mapping, glycan profiling, LC-MS, LC-fluorescence, MALDI MS     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.     

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.     

农作物虫害的机器检测与监测技术研究进展

在早期发现并准确定位害虫, 对其未来的发展趋势作出评价, 可提高施药处方决策和综合防治的针对性和准确性。在作物虫害信息的获取中, 传统的检测和监测方法不但耗时、费力, 而且导致的预报滞后会进一步增加损失程度, 很难较好地满足现代农业的精准生产要求。本文介绍了国内外学者在田间作物上开展害虫及其危害状况的机器检测和监测技术研究取得的进展, 包括声特征检测法、雷达观测法、图像识别法以及光谱监测法等, 讨论了现有技术的局限性, 指出了未来作物虫害机器检测和监测技术的可能发展方向是采用多种技术相结合的组合式检测和监测方法, 从多个角度获取特定虫害的相关信息, 相互进行实证检验, 以提高作物虫害机器检测和监测的精度及效率。     

基于植物重组蛋白产量提高的研究进展

重组蛋白为疾病治疗提供了新手段,同时创造了可观的经济效益。利用经济作物(主要是烟草)、谷类作物、豆科作物和蔬菜作物生产具有药用价值的重组蛋白是“分子农业”最热门的研究内容。尽管许多重组蛋白已在植物中表达,但只有一小部分已成功投入使用。为了极大地克服限制植物生产重组蛋白发展的问题,研究人员改进表达系统以增加重组蛋白的产量。本文从分析植物产生重组蛋白产量低和/或生物活性低等问题入手,综述了近些年来解决这些问题的优化策略,同时提出了提高植物生产重组蛋白产量的研究方向。     

Advanced oxidation protein products and inflammatory markers in liver cirrhosis: a comparison between alcohol-related and HCV-related cirrhosis

Advanced oxidation protein products (AOPPs) are protein markers of oxidative stress with pro-inflammatory properties that accumulated in liver cirrhosis. In the present study, we investigated the association between chronic inflammatory response triggered by AOPPs and the severity of liver disease as assessed by the Child-Pugh score. Plasma concentrations of AOPPs and inflammatory markers such as C-reactive protein, tumor necrosis factor-α, and interleukin-6 were measured in 41 patients with HCV-related cirrhosis, 43 patients with alcohol-related liver cirrhosis (ALC), and in 30 age and sex matched controls. In comparison with controls, AOPPs were increased in HCV-related compensated (Child-Pugh A) and decompensated (Child-Pugh B-C) cirrhosis and in alcohol-related compensated cirrhosis. AOPPs level positively correlated with Child-Pugh score in alcohol-related cirrhosis but not in HCV-related cirrhosis and the correlation with the indices of chronic inflammation was stronger in ALC. In turn, AOPPs in HCV-related cirrhosis was related to inflammation to a lesser extent, but a significant correlation with antioxidant defense could be noted. In summary, liver cirrhosis was associated with increased formation of AOPPs, which differed between alcohol-related and HCV-related cirrhosis with respect to the relationship between AOPPs and antioxidant defense, stage of liver cirrhosis, and inflammatory response. The significant correlation between AOPPs accumulation and indices of chronic inflammation, more specifically TNF-α, suggests that oxidative stress may be a mediator of chronic inflammatory state in the early stage of alcohol-related cirrhosis.     

Plant pathology: monitoring a pathogen-targeted host protein

A plant protein RIN4 is targeted and modified by bacterial pathogens as part of the disease process. At least two host resistance proteins monitor this pathogen interference and trigger the plant's defence responses.     

腺病毒载体介导的重组蛋白sTNFRII-gAD快速表达和制备

利用腺病毒载体高效感染哺乳动物细胞及表达外源基因的特性,建立一种快速高效表达及制备重组蛋白的方法,并用该方法获得sTNFRII-gAD (可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白) 蛋白纯品。首先用携带EGFP基因的腺病毒载体rAd5-EGFP以不同的腺病毒用量 (MOI) (0~1 000) 感染BHK21c022细胞,观察比较其转导效率和细胞毒性。用AdMax腺病毒载体系统制备携带融合基因sTNFRII-gAD的重组复制缺陷型5型腺病毒rAd5-sTNFRII-gAD。用rAd5-sTNFRII-gAD以不同的MOI (0~1 000) 感染BHK21c022细胞,收取上清进行Western blotting分析,比较上清中sTNFRII-gAD蛋白的表达量。在此基础上,用rAd5-sTNFRII-gAD以MOI 100感染大量培养的BHK21c022细胞,在无血清培养条件下反复多次收取培养上清,经过硫酸铵浓缩、分子筛柱层析、透析等步骤浓缩和纯化sTNFRII-gAD融合蛋白,并体外测定该融合蛋白拮抗TNFa的活性。结果获得了携带sTNFRII-gAD融合基因的重组腺病毒rAd5-sTNFRII-gAD;用rAd5-EGFP感染BHK21c022细胞结果表明,随着腺病毒用量的增高,表达EGFP蛋白的BHK21c022细胞数量和亮度明显增加;MOI在0~100之间被感染的BHK21c022细胞未表现出明显的细胞毒性,MOI为1 000时可观察到细胞变圆和少量死亡现象。Western blotting分析结果表明,随着腺病毒用量的增高,培养上清中sTNFRII-gAD融合蛋白表达量明显增加,以MOI为1 000时最高。在此基础上,我们用MOI为100的rAd5-sTNFRII-gAD感染5个转瓶培养的BHK21c022细胞以制备sTNFRII-gAD融合蛋白。每个转瓶加无血清培养液100 mL,每48 h收获上清1次并换液,反复6次收取培养上清共约3 L,经过纯化获得了约11 mg的sTNFRII-gAD融合蛋白。体外活性测定实验表明,获得的sTNFRII-gAD融合蛋白能有效拮抗TNFα对L929细胞的杀伤作用。腺病毒载体/BHK21细胞表达系统是一个简便、高效、通用的表达系统,利用该系统成功制备了有生物学活性的sTNFRII-gAD融合蛋白。该表达系统的特点是生产规模易于放大,适应无血清培养,可以反复多次收获目标蛋白。