Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography

GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.     

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm³ after 3 days, 1.24 ± 0.76 mm³ after 7 days, 0.33 ± 0.03 mm³ after 14 days, and 0.09 ± 0.05 mm³ after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.     

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

Purification of GFP fusion proteins from transgenic plant cell cultures

Green fluorescence protein (GFP) has become a widely used reporter in many areas of life science. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. GFP itself has been purified from recombinant organisms by several methods, often involving unfavorable conditions (e.g., use of organic solvents and/or low pH) that may be destabilizing to some proteins. In this study, we have developed a general recovery scheme that entails a simple three-step purification procedure for GFP fusion proteins produced in tobacco suspension cells, with the intent of maximizing purity and yield under gentle conditions so as to maintain the integrity of the fusion partner. Ammonium sulfate treatment at 30% (v/v) precipitated particulate matter and removed aggregated material while simultaneously maintaining GFP solubility and increasing hydrophobicity. Hydrophobic interaction chromatography was then performed to eliminate the majority of background proteins while eluting GFP and fusions in a low ionic buffer suitable to be directly applied to an ion-exchange column as the final step. Three intracellular proteins, secreted alkaline phosphatase (SEAP), and granulocyte-macrophage colony-stimulating factor (GMCSF), each fused to GFP, as well as GFP itself, were recovered with yields exceeding 70% and purity levels over 80%. This purification scheme exploits the hydrophobic nature of GFP while maintaining a gentle environment for labile fusion partners. Although some optimization may be required, we believe this scheme may serve as a benchmark for purifying other GFP fusion proteins.     

Development of a bicistronic expression system in the branchiopod crustacean Daphnia magna

The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna—an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B‐ green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B‐GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A‐mediated bicistronic expression would be a promising tool for evo‐devo studies of this species.     

Using electroporation and lipid-mediated transfection of GFP-expressing plasmids to label embryonic avian cells for vital confocal and two-photon microscopy

Fluorescent proteins have emerged as an ideal fluorescent marker for studying cell morphologies in vital systems. These proteins were first applied in whole organisms with established germ-line transformation protocols, but now it is possible to label cells with fluorescent proteins in other organisms. Here we present two ways to introduce GFP expressing plasmids into avian embryos for vital confocal and two-photon imaging. First, electroporation is a powerful approach to introduce GFP into the developing neural tube, offering several advantages over dye labeling. Second, we introduce a new lipid-based transfection system for introducing plasmid DNA directly to a small group of injected cells within live, whole embryos. These complementary approaches make it possible to transfect a wide-range of cell types in the avian embryo and the bright, stable, uniform expression of GFP offers great advantages for vital fluorescence imaging.     

Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 “long” (M1) and “short” (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 °C indicated that OAPs diffuse slowly (D ∼ 10⁻¹² cm²/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.     

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.     

An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability

Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.     

Assessment of membrane protein expression and stability using a split green fluorescent protein reporter

Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP(11)) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP(1-10)). The signal obtained after sequential expression of SteT (l-serine/l-threonine exchanger of Bacillus subtilis) fused to GFP(11) followed by GFP(1-10) specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT-GFP(11) with purified GFP(1-10) was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT-GFP(11) following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.     

��Pix Up-regulates Na+/H+ Exchanger 3 through a Shank2-mediated Protein-Protein Interaction

Na⁺/H⁺ exchanger 3 (NHE3) plays an important role in neutral Na⁺ transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and βPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.     

Use of Green Fluorescent Protein in mouse embryos

Green Fluorescent Protein (GFP) has rapidly been established as a versatile and powerful cell marker in many organisms. Initial problems in using it in mammalian cells were solved by introducing mutations to increase its solubility at higher temperatures, such that GFP has now been used as a reporter in both gene expression and cell lineage studies, and to localize proteins within mammalian cells. GFP has two unique advantages: (i) the protein becomes fluorescent in an autocatalytic reaction, so that it can be introduced into any cell type simply as a cDNA or mRNA, or as protein; (ii) it is "bright" enough to be visualized in living cells under conditions that do not cause photodamage to the cells. In this article we outline the ways in which we have used GFP mRNA and cDNA in our studies of mouse cell lineages, and to characterize the behavior of proteins within the embryos.     

Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins

Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.     

Functional expression of green fluorescent protein derivatives in Halobacterium salinarum

We investigated the applicability of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for gene expression in an extremely halophilic organism: Halobacterium salinarum. Two recombinant GFPs were fused with bacteriorhodopsin, a typical membrane protein of H. salinarum. These fusion proteins preserved the intrinsic functions of each component, bacteriorhodopsin and GFP, were expressed in H. salinarum under conditions with an extremely high salt concentration, and were proved to be properly localized in its plasma membrane. These results suggest that GFP could be used as a versatile reporter of gene expression in H. salinarum for investigations of various halophilic membrane proteins, such as sensory rhodopsin or phoborhodopsin.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

The cadA gene in Dictyostelium encodes a Ca²⁺-dependent cell adhesion molecule DdCAD-1 that contains two β-sandwich domains. DdCAD-1 is synthesized in the cytoplasm as a soluble protein and then transported by contractile vacuoles to the plasma membrane for surface presentation or secretion. DdCAD-1-green fluorescent protein (GFP) fusion protein was expressed in cadA-null cells for further investigation of this unconventional protein transport pathway. Both morphological and biochemical characterizations showed that DdCAD-1-GFP was imported into contractile vacuoles. Time-lapse microscopy of transfectants revealed the transient appearance of DdCAD-1-GFP-filled vesicular structures in the lumen of contractile vacuoles, suggesting that DdCAD-1 could be imported by invagination of contractile vacuole membrane. To assess the structural requirements in this transport process, the N-terminal and C-terminal domains of DdCAD-1 were expressed separately in cells as GFP fusion proteins. Both fusion proteins failed to enter the contractile vacuole, suggesting that the integrity of DdCAD-1 is required for import. Such a requirement was also observed in in vitro reconstitution assays using His₆-tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca²⁺-binding sites were mutated, indicating a role for Ca²⁺ in the import process. Spectral analysis showed that mutations in the Ca²⁺-binding sites resulted in subtle conformational changes. Indeed, proteins with altered conformation failed to enter the contractile vacuole, suggesting that the import signal is somehow integrated in the three-dimensional structure of DdCAD-1.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91^phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91^phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91^phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91^phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.     

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp , produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfp s as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.