Understanding the role of Arg96 in structure and stability of green fluorescent protein

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide-induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5- and 50-times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl-induced unfolding both in kinetics and in the quasi-equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP.     

Visualizing the complexity of proteins in living cells with genetic code expansion

Genetic code expansion has emerged as an enabling tool to provide insight into functions of understudied proteinogenic species, such as small proteins and peptides, and to probe protein biophysics in the cellular context. Here, we discuss recent technical advances and applications of genetic code expansion in cellular imaging of complex mammalian protein species, along with considerations and challenges on using the method.     

Visualization of molecular and cellular events with green fluorescent proteins in developing embryos: a review

An Erratum has been published for this article in Luminescence (2003) 18(4) 243 During the past 5 years, green fluorescent protein (GFP) has become one of the most widely used in vivo protein markers for studying a number of different molecular processes during development, such as promoter activation, gene expression, protein trafficking and cell lineage determination. GFP fluorescence allows observation of dynamic developmental processes in real time, in both transiently and stably transformed cells, as well as in live embryos. In this review, we include the most up‐to‐date use of GFP during embryonic development and point out the unique contribution of GFP visualization, which resulted in novel discoveries. Copyright © 2002 John Wiley & Sons, Ltd.     

橙色荧光蛋白——绿色荧光蛋白GFPxm的改造

最近报道了从大型多管水母中分离出新的gfp基因。经大肠杆菌表达并纯化出的绿色荧光蛋白 (GFPxm)具有 4 76nm的激发峰和 4 96nm的发射峰 ,但是只能在低温下成熟的缺点限制了它的应用。这里进一步报道GFPxm的 12种突变型。在大肠杆菌中的表达结果表明 ,有 7种突变型在 37℃条件下产生高的荧光强度。在 2 5、32和 37℃条件下表达 6h ,GFPxm16、GFPxm18和GFPxm19的相对荧光强度均高于增强型绿色荧光蛋白 (EGFP) ,而GFPxm16和GFPxm16 3在 4 2℃高温表达时仍能保持高的荧光强度。这 7种突变型中的 4种在哺乳动物细胞中已获得良好表达。此外 ,有 6种突变型的荧光光谱红移 ,目前所达到的最长激发峰为 5 14nm、最长发射峰为 5 2 5nm。另外有 3种突变型具有包括紫外在内的两个激发峰 ,1种突变型只有单一的紫外激发峰。首次报道具有橙色荧光的突变型OFPxm ,它的激发峰为 5 0 9nm、发射峰为 5 2 3nm。 5 2 3nm属于黄绿色 ,但肉眼看到的蛋白为橙色。OFPxm在高温下可得到高水平表达且很好地成熟 ,但是因为低的量子产率而荧光强度相对较低。     

绿色荧光蛋白——照亮生命科学的一盏明灯

绿色荧光蛋白的发现及应用具有划时代的重要意义,它不仅为当代生物学研究提供了极为实用的基本研究手段,并且在此基础上改造发展和发现了一系列荧光蛋白,拓展了应用范围。这使得对微观生物学的研究也可以进入一个时空结合,研究鲜活动态过程的新时代。本文主要回顾总结了绿色荧光蛋白的发现、优化改造及其应用。     

The ability of green fluorescent proteins for photoconversion under oxygen-free conditions is determined by the chromophore structure rather than its amino acid environment

Photoconversion of various green and cyan fluorescent proteins to the red fluorescent state under the oxygen-free conditions was studied. Such photoconversion has earlier been described for the EGFP green fluorescent protein. Phylogenetically distant fluorescent proteins that have a low identity of their amino acid sequences but contain chemically identical chromophores based on a Tyr residue were shown to be susceptible to this type of photoconversion. At the same time, the ECFP protein, which has 92% homology with EGFP but contains a chromophore based on tryptophan did not undergo the photoconversion. Thus, it is precisely the chromophore structure, rather than the amino acid environment that determines the ability of green fluorescent proteins to display photoconversion to the red fluorescent state under anaerobic conditions.     

Chromoproteins of the green fluorescent protein family: Properties and applications

The distribution in nature and the spectral and structural properties of chromoproteins of the green fluorescent protein (GFP) family and their differences from one another and other fluorescent proteins of this family are considered. Discussed in detail are practical applications of the chromoproteins and their mutant variants that have unique characteristics not found among natural proteins of the GFP family, such as far-red or photoconvertible fluorescence, a large Stokes shift, enhanced phototoxicity, etc.     

Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants

Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.     

Evolutionary and functional diversity of green fluorescent proteins in cephalochordates

Green fluorescent protein (GFP) has been widely used as a molecular marker in modern biological research. Before the recent report of one GFP gene in Branchiostoma floridae, GFP family members were cloned only from other two groups of species: Cnidaria and Copepoda. Here we describe the complete GFP gene repertoire of B. floridae which includes 13 functional genes and 2 pseudogenes, representing the largest GFP family found so far. Coupling with nine other GFP sequences from another two species of genus Branchiostoma and the sequences from Cnidaria and Copepoda, we made a deep-level phylogenetic analysis for GFP genes in cephalochordates and found: 1) GFP genes have experienced a divergent evolution in cephalochordates; 2) all amphioxus GFP genes form four main clades on the tree which had diverged before the radiation of the last common ancestor of all extant cephalochordates; 3) GFP genes in amphioxus shared a common ancestor with that in Copepoda rather than being derived from horizontal gene transfer, which indicates that our ancestor was derived from a fluorescent organism and lost this ability after its separation from Cephalochordata, and also makes GFP a rare gene which has a rather unusual evolutionary path. In addition, we also provided evidence indicating that GFP genes have evolved divergent functions by specializing their expression profile, and different fluorescent spectra by changing their emission peaks. These findings spark two interesting issues: what are GFP in vivo functions in cephalochordates and why they are lost in other examined deuterostomes?     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

根癌农杆菌介导的增强型绿色荧光蛋白基因在淡紫拟青霉中的转化

为了实现增强型绿色荧光蛋白基因 (egfp) 在生防真菌淡紫拟青霉9410菌株中的转化,借助中间质粒pcDNA3.1(-) 构建nptⅡ-egfp融合基因的表达载体pUPNGT,然后采用根癌农杆菌介导的转化法将egfp基因转化到淡紫拟青霉9410菌株中。PCR检测和Southern blotting分析结果表明,egfp基因以单拷贝形式整合到淡紫拟青霉9410的基因组中。荧光显微镜观察结果显示,转化子在488 nm下能产生绿色荧光。这些结果说明egfp基因已成功转化至淡紫拟青霉9410菌株并获得表达。这些工作可为淡紫拟青霉在不同条件下的防效评价、环境安全评价等提供新的途径和方法。     

Differentiation of embryonic stem cell to astrocytes visualized by green fluorescent protein

Green fluorescent protein (GFP) gene was transfected and expressed in murine embryonic stem (ES) cells under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Stably transfected cells were characterized by immunohistochemistry and by fluorescence microscopy. Cells containing GFP were differentiated to Type I and Type II astrocytes after induction by all-trans retinoic acid. Differentiated cells were expressed GFP and visualized by fluorescence microscopy. Differentiated cells expressed GFP were correlated with the expression of GFAP and morphological change. It demonstrates that the cell line expressed GFP can be used to trace the morphological changes of astrocytes during differentiation, and further for the isolation of astrocytes from the mixed cells differentiated from ES cell.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

Effect of polyethylene glycol on the thermal stability of green fluorescent protein

Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70–95°C. Thermal stability was expressed in decimal reduction time. It was observed that the D‐values obtained were almost constant for temperatures of 85, 90, and 95°C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75°C, PEG 600 and 4,000 g/mol stabilized GFP. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Soluble,highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants

Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is brighter because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.     

绿色荧光蛋白GFP基因与新生隐球菌荚膜基因的融合表达系统

目的构建新生隐球菌荚膜基因与绿色荧光蛋白的融合表达系统。方法PCR法扩增CAP60基因片段,测序验证其准确性。将其与多个必需基因共同连人穿梭质粒。结果获得6150bps大小的质粒,该质粒含有荚膜基因启动子、终止子及荧光蛋白的基因。结论将新生隐球菌荚膜基因与荧光蛋白基因融合表达,将会有利于对荚膜的生化合成途径作进一步研究。     

Stability of the genetic code and optimal parameters of amino acids

The standard genetic code is known to be much more efficient in minimizing adverse effects of misreading errors and one-point mutations in comparison with a random code having the same structure, i.e. the same number of codons coding for each particular amino acid. We study the inverse problem, how the code structure affects the optimal physico-chemical parameters of amino acids ensuring the highest stability of the genetic code. It is shown that the choice of two or more amino acids with given properties determines unambiguously all the others. In this sense the code structure determines strictly the optimal parameters of amino acids or the corresponding scales may be derived directly from the genetic code. In the code with the structure of the standard genetic code the resulting values for hydrophobicity obtained in the scheme “leave one out” and in the scheme with fixed maximum and minimum parameters correlate significantly with the natural scale. The comparison of the optimal and natural parameters allows assessing relative impact of physico-chemical and error-minimization factors during evolution of the genetic code. As the resulting optimal scale depends on the choice of amino acids with given parameters, the technique can also be applied to testing various scenarios of the code evolution with increasing number of codified amino acids. Our results indicate the co-evolution of the genetic code and physico-chemical properties of recruited amino acids.     

Applications of Genetic Code Expansion in Studying Protein Post-translational Modification

Various post-translational modifications can naturally occur on proteins, regulating the activity, subcellular localization, interaction, or stability of the proteins. However, it can be challenging to decipher the biological implication or physiological roles of site-specific modifications due to their dynamic and sub-stoichiometric nature. Genetic code expansion method, relying on an orthogonal aminoacyl-tRNA synthetase/tRNA pair, enables site-specific incorporation of non-canonical amino acids. Here we focus on the application of genetic code expansion to study site-specific protein post-translational modification in vitro and in vivo. After a brief introduction, we discuss possibilities of incorporating non-canonical amino acids containing post-translational modifications or their mimics into target proteins. This approach is applicable for Ser/Thr/Tyr phosphorylation, Tyr sulfation/nitration/hydroxylation, Lys acetylation/acylation, Lys/His mono-methylation, as well as Arg citrullination. The next section describes the use of a precursor non-canonical amino acid followed by chemical and/or enzymatic reactions to afford the desired modification, such as Cys/Lys acylation, ubiquitin and ubiquitin-like modifications, as well as Lys/Gln methylation. We also discuss means for functional regulation of enzymes involving in post-translational modifications through genetically incorporated non-canonical amino acids. Lastly, the limitations and perspectives of genetic code expansion in studying protein post-translational modification are described.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.     

Concentrating rotavirus and recombinant-enhanced green fluorescent protein from E. coli using a pH-sensitive hydrogel

A rotavirus and a recombinant-enhanced green fluorescent protein from E. coli were concentrated 1.7 times and 1.5 times, respectively, by ultrafiltration at 37°C and pH 7 using a pH-sensitive hydrogel, poly(N-isopropylacrylamide-co-N, N-dimethylaminopropyl methacrylamide). Recoveries were 77% and 69%, respectively, and separation efficiencies were 58% and 44%, respectively. The concentration increase of the protein was confirmed by SDS-PAGE.