Recombinant Fluorescent Proteins Research Articles

Binding of Calcineurin Homologous Protein Isoforms 1 and 2 for the Na/H+ Exchanger Isoform 1 Using Recombinant Fluorescent Proteins

The sodium hydrogen exchanger isoform 1 (NHE1) is a transmembrane transport protein critical for cell motility and intracellular pH regulation. NHE1 is regulated by a variety of cofactors and binding partners, including calcineurin homologous protein (CHP). Isoform 1 of CHP (CHP1) is expressed ubiquitously and is required for basal NHE1 activity; however, isoform 2 (CHP2) expression is only detected in transformed cancer cells and has been implicated as a putative target for cancer therapies. We have expressed and purified recombinant‐affinity tagged c‐terminal tail of NHE, RFP‐CHP1 and GFP‐CHP2 and used these for in vitro capture assays. This work demonstrates the affinity of each CHP isoform with NHE1. In vivo, we have stably expressed full‐length GST‐tagged NHE1 in lung fibroblast cells (PS120). Using pull‐down assays in cells transiently transfected with CHP1 or CHP2 we further demonstrate the affinity of CHP1 and CHP2 with NHE1. Follow‐up experiments with altered NHE1 amino acids are used to define potential sites of interaction of CHP2 and NHE1. Together this data illustrates the different affinity of CHP2 for NHE1 binding and points to a potential regulatory site for NHE1 function.

Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases

We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.

Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems.

BackgroundNew high-throughput screening systems for microbial systems, e.g. the BioLector technology, are simple to handle and offer various options of optical online measurements. The parallelization and small scale in microtiter plates allow economical high throughput and, hence, to screen many parameters in reasonable time. Fluorescent proteins as fluorescent tags made the tracking of cellular proteins in-vivo a routine task. All these tools significantly contribute to the understanding of bioprocesses. But, there are some pitfalls which might mislead the user of such techniques.ResultsIn this work the bacterium E. coli and the yeast K. lactis expressing the recombinant fluorescent proteins GFP, YFP, FbFP and mCherry were investigated. Cultivations were performed applying special microtiter plates with optodes for dissolved oxygen tension (DOT) and pH measurement in the BioLector system. In this way, microbial growth, protein formation, DOT and pH were monitored on-line via optical signals. During these studies it became obvious that fluorescent proteins can interfere with the optical signals leading to incorrect results. In this work these effects are characterized in detail and possibilities are presented how such adverse effects can be corrected or minimized by mathematical procedures or modification of the measuring method. Additionally, it is shown that morphological changes of cells can affect the biomass on-line monitoring via scattered light.ConclusionsThe here reported phenomena refer to typical experiments in biotechnological labs. For this reason these aspects are highlighted in this work to make operators of such valuable techniques as the BioLector aware for potential pitfalls and resulting misinterpretations. With the right approach it is possible to minimize existing problems and deal with them.

FRETting for a benchmark (LB280)

Fluorescence resonance energy transfer (FRET) is a powerful tool to study molecular interactions, especially protein‐protein interactions (PPI). Recombinant fluorescent proteins enable the study of such interaction in living cells. However, there is no reference study yet detailing the limits of the strength of molecular interactions that can be detected by live‐cell FRET. Using a test set of 11 pairs of small interacting proteins for whom the respective affinities (Kd, dissociation constant) range between mM ‐ nM, we establish the limits with live‐cell microscopy. This study benchmarks intracellular FRET and is a very useful reference for other intracellular PPI methods such as BiFC (Bimolecular Fluorescence Complementation) and IDPRIME (Interaction‐Dependent Probe Incorporation Mediated by Enzymes).Grant Funding Source: BCAAP (Biological Control and Analysis through Applied Photonics) and AES (Agricultural Experiment Station) of SDSU

High‐throughput screening for transglutaminase activities using recombinant fluorescent proteins

Since detailed evaluation of specific transglutaminases (TGs) from various species requires identification of their substrate specificities, rapid substrate screening method by measurement of their relative activities is in great demand. Here, a novel evaluation method of TG activity was developed using two recombinant fluorescent proteins (FPs), that is, eYFP and DsRed, tagged with TG substrate peptides. By cross-linking the two FPs based on the tagged target peptide sequences at their C-terminus, the expression of co-transformed TG allows quenching of the yellow fluorescence intensities. It was shown that the degree of in vivo fluorescent quenching by the TG activity agrees well with its in vitro reaction data, suggesting that this system can be used to identify relative substrate specificity of TGs for target peptide sequences. Using this method, the lysine substrates of TGs from Bacillus species (BTG) were evaluated, and the newly selected pentapeptide, KTKTN showed almost the same reactivity with the well-known hexa-lysine (K₆) substrate for BTG reaction.

Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death.

IntroductionThe worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors.Specific aimThe specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases).MethodsThe DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers’ bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies’ guided DNA vectors delivered the transgenes for the human recombinant DNases’ into proliferating stem cells.ResultsDifferentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells’ genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die.ConclusionHerein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy.

Luciferase and Bioluminescence Microscopy for Analyses of Membrane Dynamics in Living Cells

Luciferase-based bioluminescence is a technique used for non-invasive analysis of molecular functions in living cells. New recombinant luciferase and advanced CCD (Charge Coupled Device) cameras allow the detection of bioluminescent signals at submicron resolution. These recombinant luciferase proteins have the potential to facilitate great advances in the study of membrane dynamics at both the subcellular and whole organism levels. Bioluminescence is the emission of light generated by the enzymatic catalysis of a chemical substrate. This is different than fluorescence which is the emission of light generated by the absorption of photons. While the brightness of a fluorescent body correlates with its molar extinct coefficient (ability of a molecule to absorb photons) and quantum yield (ability of a molecule to emit photons), brightness of bioluminescent body correlates with a concentration of the substrate and efficiency of the bioluminescent enzyme in catalyzing the substrate. Fluorescence microscopy is widely used to analyse functions of molecules in living cells. Genetically engineered fluorescent proteins that are fused to a protein of interest via recombinant DNA techniques can determine subcellular localization of a protein of interest. From a membrane science and technology point of view, recombinant fluorescent proteins have been used to study membrane-associated processes in living cells such as endocytosis [1], morphological changes of the Golgi complex [2], and protein clusters in the membrane [3]. It is difficult to use fluorescent proteins for in vivo analyses because it is difficult for the excitation light to reach a fluorescent protein located in cells buried deep within a tissue or organism. Moreover, background fluorescence is relatively high because many tissues contain molecules that naturally fluorescence. Also, the working distance of most compound microscope objectives is very small (millimeters). Therefore, the use of fluorescent proteins in living cells is restricted to in vitro experiments except in special cases [4,5].

Universal and rapid method for purification of GFP-like proteins by the ethanol extraction

GFP-like fluorescent proteins (FPs) are crucial in biological and biomedical studies. The majority of FP purification techniques either include multiple time-consuming chromatography steps with a low yield of the desired product or require prior protein modification (addition of special tags). In the present work, we propose an alternative ethanol extraction-based technique previously used for GFP purification and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purification of any amount of protein and requires no expensive reagents and equipment.

Reporting neural activity with genetically encoded calcium indicators.

Genetically encoded calcium indicators (GECIs), based on recombinant fluorescent proteins, have been engineered to observe calcium transients in living cells and organisms. Through observation of calcium, these indicators also report neural activity. We review progress in GECI construction and application, particularly toward in vivo monitoring of sparse action potentials (APs). We summarize the extrinsic and intrinsic factors that influence GECI performance. A simple model of GECI response to AP firing demonstrates the relative significance of these factors. We recommend a standardized protocol for evaluating GECIs in a physiologically relevant context. A potential method of simultaneous optical control and recording of neuronal circuits is presented.

Selection of Genetically Encoded Fluorescent Single Domain Antibodies Engineered for Efficient Expression in Escherichia coli

Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology and proteomics. However, the efficient production of single chain antibodies fused to fluorescent proteins like green fluorescent protein is still a major challenge. This is due to the opposite redox requirements of recombinant antibodies and fluorescent proteins for proper folding. To overcome this obstacle, we designed a novel strategy to directly select functional fluorescent antibodies (fluobodies) engineered for efficient cytosolic expression in Escherichia coli, starting from a llama heavy chain antibody (VHH) library. We used this strategy to produce several VHH fluobodies directed against microtubules. After biochemical characterization in vitro, we used a selected fluobody as a convenient biomarker of the microtubule cytoskeleton in eukaryotic cells and engineered its properties. This work provides a reliable approach for the production of fluobodies in wild-type E. coli and furthermore suggests that fusions of recombinant VHH with other cytosolic proteins are now possible, creating new opportunities for biotechnology and biomedical research.

Cover Picture: In Vivo Screening Identifies a Highly Folded β‐Hairpin Peptide with a Structured Extension (ChemBioChem 8/2007)

The cover picture shows the NMR‐derived structure of a β‐hairpin peptide that was identified through a fluorescence‐based screen in live cells. A Petri dish covered in colonies of E. coli expressing recombinant fluorescent proteins was illuminated by ultraviolet light. Each bacterial colony expresses a single protein composed of a tandem fusion of a cyan (CFP) and a yellow (YFP) fluorescent protein. As shown in ribbon representations, peptide sequences are fused between these CFP and YFP units. Peptides that fold into β‐hairpin structures tend to bring the two fluorescent proteins into closer proximity, and thus a higher FRET signal is observed. Residues in the green portions of the peptides were randomized to create libraries of many hundreds of variants, and the peptide sequence that provided the highest FRET efficiency was identified by FRET‐based screening of bacterial colonies. The 3D structure of this peptide revealed that the selected residues participated in a cross‐strand cation–π interaction that held the ends of the peptide closer together. More details can be found in the article by Robert E. Campbell et al. on p. 880 ff. (Image credit: Annie Tykwinski and R.E.C.)

Functional Characterization of cis and trans Activity of the Flavivirus NS2B-NS3 Protease

Flaviviruses are serious human pathogens for which treatments are generally lacking. The proteolytic maturation of the 375-kDa viral polyprotein is one target for antiviral development. The flavivirus serine protease consists of the N-terminal domain of the multifunctional nonstructural protein 3 (NS3) and an essential 40-residue cofactor (NS2B(40)) within viral protein NS2B. The NS2B-NS3 protease is responsible for all cytoplasmic cleavage events in viral polyprotein maturation. This study describes the first biochemical characterization of flavivirus protease activity using full-length NS3. Recombinant proteases were created by fusion of West Nile virus (WNV) NS2B(40) to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg(459) decreasing Gly(460) within the C-terminal helicase region of NS3 was cleaved more slowly. Autolytic cleavage reactions also occurred in NS2B-NS3 recombinant proteins from yellow fever virus, dengue virus types 2 and 4, and Japanese encephalitis virus. Cis and trans cleavages were distinguished using a noncleavable WNV protease variant and two types of substrates as follows: an inactive variant of recombinant WNV NS2B-NS3, and cyan and yellow fluorescent proteins fused by a dodecamer peptide encompassing a natural cleavage site. With these materials, the autolytic cleavages were found to be intramolecular only. Autolytic cleavage of the helicase site was insensitive to protein dilution, confirming that autolysis is intramolecular. Formation of an active protease was found to require neither cleavage of NS2B from NS3 nor a free NS3 N terminus. Evidence was also obtained for product inhibition of the protease by the cleaved C terminus of NS2B.

Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates.

Ligand binding and luciferase interaction properties of the recombinant protein corresponding to the lumazine protein gene (EMBL X56534) of Photobacterium leiognathi have been determined by fluorescence dynamics, circular dichroism, gel filtration, and SDS-PAGE. Scatchard analysis of a fluorescence titration shows that the apoprotein possess one binding site, and at 30 degrees C the KdS (microM) are as follows: 6,7-dimethyl-8-ribityllumazine, 0.26; riboflavin, 0.53; and much more weakly bound FMN, 30. All holoproteins are highly fluorescent and have absorption spectra distinct from each other and from the free ligands. The longest wavelength absorption maxima are, respectively (nm, 2 degrees C), 420, 463, and 458. Ligand binding produces no change in the far-UV circular dichroism; all have mean residual ellipticity at 210 nm of -6500 deg cm2 dmol-1, the same as the native protein. However, in the bioluminescence reaction only the lumazine holoprotein shows a bioluminescence effect. Fluorescence emission anisotropy decay was used to establish that none of these holoproteins complexed with native luciferase and that the lumazine protein alone formed a 1:1 complex with the luciferase hydroxyflavin fluorescent transient and the luciferase peroxyflavin intermediates, revealed by a dominant channel of anisotropy loss, with rotational correlation time of 2.5 ns, and attributed to excitation transfer from the luciferase flavin donor to the acceptor, the lumazine ligand. The complex stability was sufficient to allow its isolation by FPLC gel filtration and verification by SDS-PAGE. These methods also confirmed the absence of interaction of the holoflavoproteins.