Production and single-step purification of EGFP and a biotinylated version of the Human Rhinovirus 14 3C protease

The fluorescent reporter enhanced Green Fluorescent Protein (EGFP) has been used for assaying a wide range of biological activities ranging from gene expression, or localization of target proteins through to intermolecular interactions. However, over-production of this protein in Escherichia coli has resulted in the presence of inclusion bodies, which complicates recovery of the protein in significant quantities. In this paper, we describe a single-step method for isolating the protein from a Glutathione-S-Transferase (GST) fusion protein, release of the EGFP protein from the fusion was demonstrated using a biotinylated variant of Human Rhinovirus 14 3C protease that we have also constructed. We also suggest the potential uses of the biotinylated protease for bionanotechnology and synthetic biology.     

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.     

Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells

Fluorescent proteins have become an invaluable tool in cell biology. The green fluorescent protein variant EGFP is especially widely applied. Use of fluorescent proteins, including EGFP, however can be hindered by inefficient protein folding, resulting in protein aggregation and reduced fluorescence. This is especially profound in prokaryotic cells. Furthermore, EBFP, a blue fluorescent variant of EGFP, is rarely used because of its dim fluorescence and fast photobleaching. Thus, efforts to improve properties such as protein folding, fluorescence brightness, and photostability are important. Strongly enhanced green fluorescent (SGFP2) and strongly enhanced blue fluorescent (SBFP2) proteins were created, based on EGFP and EBFP, respectively. We used site-directed mutagenesis to introduce several mutations, which were recently shown to improve the fluorescent proteins EYFP and ECFP. SGFP2 and SBFP2 exhibit faster and more efficient protein folding and accelerated chromophore oxidation in vitro. For both strongly enhanced fluorescent proteins, the photostability was improved 2-fold and the quantum yield of SBFP2 was increased 3-fold. The improved folding efficiency reduced the extent of protein aggregation in Escherichia coli, thereby increasing the brightness of bacteria expressing SGFP2 7-fold compared to the brightness of those expressing EGFP. Bacteria expressing SBFP2 were 16-fold more fluorescent than those expressing EBFP. In mammalian cells, the improvements were less pronounced. Cells expressing SGFP2 were 1.7-fold brighter than those expressing EGFP, which was apparently due to more efficient protein expression and/or chromophore maturation. Mammalian cells expressing SBFP2 were 3.7-fold brighter than cells expressing EBFP. This increase in brightness closely resembled the increase in intrinsic brightness observed for the purified recombinant protein. The increased maturation efficiency and photostability of SGFP2 and SBFP2 facilitate detection and extend the maximum duration of fluorescence imaging.     

Localization of functional polypeptides in bacterial inclusion bodies

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a beta-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular beta-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.     

Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP

Background  

The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.     

Localization of Functional Polypeptides in Bacterial Inclusion Bodies

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a β-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular β-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.     

Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro

Here, we present a protocol for isolating the large N-terminal fragment of enhanced green fluorescent protein (EGFP) with a preformed chromophore. By itself, the chromophore-containing EGFP fragment exhibits very weak fluorescence, but it rapidly becomes brightly fluorescent upon complementation with the corresponding small, C-terminal EGFP fragment. Each EGFP fragment is cloned and overexpressed in E. coli as a fusion with self-splitting intein. After solubilizing and refolding these fusions from inclusion bodies, both EGFP fragments are cleaved from intein and purified using chitin columns. When these EGFP fragments are linked with the two complementary oligonucleotides and combined in equimolar amounts, fluorescence develops within a few minutes. The isolation of profluorescent protein fragments from recombinant E. coli cells requires approximately 3 d, and their conjugation to oligonucleotides requires 1-4 h.     

快速筛选高效表达重组蛋白毕赤酵母菌株新方法的建立及评价

毕赤酵母是当前应用最为广泛的重组蛋白表达系统之一,文中建立了一种快速筛选高效表达重组蛋白的毕赤酵母菌株的新方法。首先,对内质网转膜蛋白Sec63融合表达增强型绿色荧光蛋白EGFP的改造菌株GS115-E表达重组蛋白的能力进行检测;之后将携带不同拷贝数的植酸酶phy基因或木聚糖酶xyn基因的质粒转化进入GS115-E中,得到具有不同植酸酶或木聚糖酶表达水平的重组菌株,分别检测不同菌株的EGFP与重组蛋白的表达水平;最后,利用分选型流式细胞仪,根据绿色荧光值的高低对包含不同植酸酶表达水平的重组菌株的菌群进行分选。结果显示重组菌株中EGFP的荧光值与重组蛋白的活性表达水平之间具有良好的线性相关性(0.8|R|1),且利用流式细胞仪可高效地从混合菌群中筛选得到高产菌株,所分选得到的高荧光菌株在摇瓶发酵120 h时植酸酶表达水平是低荧光菌株的4.09倍。本方法通过检测菌株的EGFP荧光值代替检测重组蛋白的表达水平和活性,从而实现高表达菌株的筛选,大大提高了其应用的便捷性及通用性。与流式细胞仪、液滴微流控等高通量筛选仪器或技术结合将进一步提高筛选的速度与通量,为筛选获得高效表达重组蛋白的毕赤酵母菌株提供了简便、快速的新途径。     

Development of combinatorial bioengineering using yeast cell surface display--order-made design of cell and protein for bio-monitoring

A genetic system to display proteins as their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae, has been exploited. Surface-engineered (arming) cells displaying amylase or cellulase could assimilate starch or cellulose as the sole carbon source, although S. cerevisiae can not intrinsically assimilate them. Arming cells with a green fluorescent protein (GFP) from Aequorea victoria can emit green fluorescence from the cell surface in response to the environmental conditions. From these results, we attempted to construct a system to monitor the foreign protein production in yeast by simultaneous displaying the enhanced GFP (EGFP). The expression in yeast of the Escherichia coli beta-galactosidase-encoding gene was examined as an example of intracellular production and that of the human interferon-alpha (omega, IFN-omega)-encoding gene as an example of extracellular production. Their productions and the simultaneous surface-display of EGFP as a reporter were controlled by the same promoter, GAL1. The relationship among fluorescence signals and their productions was evaluated. The surface-display system, unlike one using tag-proteins, would be able to facilitate the monitoring of native protein productions in bioprocesses using living cells in real time by the combination of promoters and GFP variants.     

Divergent genetic control of protein solubility and conformational quality in Escherichia coli

In bacteria, protein overproduction results in the formation of inclusion bodies, sized protein aggregates showing amyloid-like properties such as seeding-driven formation, amyloid-tropic dye binding, intermolecular β-sheet architecture and cytotoxicity on mammalian cells. During protein deposition, exposed hydrophobic patches force intermolecular clustering and aggregation but these aggregation determinants coexist with properly folded stretches, exhibiting native-like secondary structure. Several reports indicate that inclusion bodies formed by different enzymes or fluorescent proteins show detectable biological activity. By using an engineered green fluorescent protein as reporter we have examined how the cell quality control distributes such active but misfolded protein species between the soluble and insoluble cell fractions and how aggregation determinants act in cells deficient in quality control functions. Most of the tested genetic deficiencies in different cytosolic chaperones and proteases (affecting DnaK, GroEL, GroES, ClpB, ClpP and Lon at different extents) resulted in much less soluble but unexpectedly more fluorescent polypeptides. The enrichment of aggregates with fluorescent species results from a dramatic inhibition of ClpP and Lon-mediated, DnaK-surveyed green fluorescent protein degradation, and it does not perturb the amyloid-like architecture of inclusion bodies. Therefore, the Escherichia coli quality control system promotes protein solubility instead of conformational quality through an overcommitted proteolysis of aggregation-prone polypeptides, irrespective of their global conformational status and biological properties.     

EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy

The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969-973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one-photon confocal and by two-photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.     

A comparative analysis of novel fluorescent proteins as reporters for gene transfer studies.

We evaluated novel fluorescent proteins (FPs) as reporters for gene transfer in animals and cells with the aim to develop more-sensitive assays for vector development and the optimization of gene transfer strategies in gene therapy. Adeno-associated virus serotype 5 vectors carrying an expression cassette with a chicken beta-actin promoter encoding the green FPs ZsGreen1, AcGFP1, hMGFP (with and without intron), and EGFP and the red FPs DsRed2 and TurboRFP were administered to mice at identical doses for each organ to target liver, lung, and muscle. Despite the fact that all FPs were expressed from an identical vector backbone, the observed number of fluorescent cells and fluorescence intensities varied between, but was consistent within, each combination of a specific protein and organ. The highest number of fluorescent cells was observed in liver with EGFP and in lung with ZsGreen1 and EGFP. In muscle, AcGFP1 and ZsGreen1 produced the most-intense fluorescence in fibers. In contrast, in culture cells, ZsGreen1 showed substantially stronger fluorescence than all other proteins. Our data demonstrate that each FP has tissue-specific expression profiles that need to be taken into consideration when comparing the performance of vectors in different organs.     

A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling

We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "Azami-Green (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric green-emitting fluorescent protein comparable to EGFP.     

Improved technique for detection of enhanced green fluorescent protein in transgenic mice.

One of the most exciting recent advances in cell biology is the possibility to use the green fluorescent protein and its various mutated forms as reporter proteins in studies carried out in vitro and in vivo. In the present study, several detection techniques for the enhanced green fluorescent protein (EGFP) were compared in transgenic mice, using fluorescence and confocal microscopy. In addition, different tissue preparation techniques (squash preparations, vibratome sections, frozen sections) were evaluated. As a model we used transgenic mice expressing EGFP under the control of a 5.0-kb fragment of the glutathione peroxidase isoenzyme 5 protein promoter (GPX5-EGFP) or under a 3.8-kb fragment of the cysteine rich protein-1 promoter (CRISP1-EGFP). In the GPX5-EGFP mice, expression of EGFP was observed in the distal part of the caput epididymis, while the CRISP1 promoter directed EGFP expression in the tubular compartment of the testis. Among the various tissue preparation procedures tested, the best morphological and histological preservation, and reproducibility in EGFP detection, were obtained using frozen sections after a slow tissue-freezing protocol developed in the present study. After slow tissue freezing, specimens of testis and epididymis could be stored at -70 degrees C for at least six weeks without any affect on EGFP fluorescence. Hence, the method developed offers the possibility to analyze EGFP fluorescence in tissues several weeks after specimen collection. The sensitivity achieved was equal to that found in immunohistochemistry, applying biotin-streptavidin-FITC detection. Confocal microscopy is known to have the advantage that fluorescence can be detected from cells in different layers. This was found to be important as regards detecting EGFP fluorescence because the fluorescence was destroyed at the cut surfaces of sections produced by either vibratome or cryomicrotome.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.     

Biological role of bacterial inclusion bodies: a model for amyloid aggregation

Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of inclusion bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make inclusion bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background.     

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.