Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background

Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.

Methodology/Principal Findings

Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.

Conclusions and Significance

The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.     

Nonlinear vibrational microscopy applied to lipid biology

Optical microscopy is an indispensable tool that is driving progress in cell biology. It still is the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Most prominently, fluorescence microscopy based on dye-labeling or protein fusions with fluorescent tags is a highly sensitive and specific method of visualizing biomolecules within sub-cellular structures. It is however severely limited by labeling artifacts, photo-bleaching and cytotoxicity of the labels. Coherent Raman Scattering (CRS) has emerged in the last decade as a new multiphoton microscopy technique suited for imaging unlabeled living cells in real time with high three-dimensional spatial resolution and chemical specificity. This technique has proven to be particularly successful in imaging unstained lipids from artificial membrane model systems, to living cells and tissues to whole organisms. In this article, we will review the experimental implementations of CRS microscopy and their application to imaging lipids. We will cover the theoretical background of linear and non-linear vibrational micro-spectroscopy necessary for the understanding of CRS microscopy. The different experimental implementations of CRS will be compared in terms of sensitivity limits and excitation and detection methods. Finally, we will provide an overview of the applications of CRS microscopy to lipid biology.     

Exogenous ether lipids predominantly target mitochondria

Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in ether lipid metabolism and intracellular ether lipid trafficking.     

An update on genetically encoded lipid biosensors

Specific lipid species play central roles in cell biology. Their presence or enrichment in individual membranes can control properties or direct protein localization and/or activity. Therefore, probes to detect and observe these lipids in intact cells are essential tools in the cell biologist’s freezer box. Herein, we discuss genetically encoded lipid biosensors, which can be expressed as fluorescent protein fusions to track lipids in living cells. We provide a state-of-the-art list of the most widely available and reliable biosensors and highlight new probes (circa 2018–2021). Notably, we focus on advances in biosensors for phosphatidylinositol, phosphatidic acid, and PI 3-kinase lipid products.     

Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.     

Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems.     

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa‐histidine tag, all attached to the C‐terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C‐ or N‐terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His₆ tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Application of the accurate mass and time tag approach in studies of the human blood lipidome

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.     

Mutants of Arabidopsis reveal many roles for membrane lipids

Polyunsaturated acyl lipids constitute approximately 50% of the hydrophobic membrane barriers that delineate the compartments of cells. The composition of these lipids is critically important for many membrane functions and, thus, for proper growth and development of all living organisms. In the model plant Arabidopsis, the isolation of mutants with altered lipid compositions has facilitated biochemical and molecular approaches to understanding lipid metabolism and membrane biogenesis. Just as importantly, the availability of a series of plant lines with specific changes in membrane lipids have provided a new resource to study the structural and adaptive roles of lipids. Now, the sequencing of the Arabidopsis genome, and the development of reverse-genetics approaches provide the tools needed to make additional discoveries about the relationships between lipid structure and membrane function in plant cells.     

Strep-tag II and Twin-Strep Based Cassettes for Protein Tagging by Homologous Recombination and Characterization of Endogenous Macromolecular Assemblies in Saccharomyces cerevisiae

Peptide sequences fused to a gene of interest facilitate the isolation of proteins or protein complexes from cell extracts. In the case of fluorescent protein tags, the tagged protein can be visually localized in living cells. To tag endogenous genes, PCR-based homologous recombination is a powerful approach used in the yeast Saccharomyces cerevisiae. This approach uses short, homologous DNA sequences that flank the tagging cassette to direct recombination. Here, we constructed a set of plasmids, whose sequences were optimized for codon usage in yeast, for Strep-tag II and Twin-Strep tagging in S. cerevisiae. Some plasmids also contain sequences encoding for a fluorescent protein followed by the purification tag. We demonstrate using the yeast pyruvate dehydrogenase (PDH) complex that these plasmids can be used to purify large protein complexes efficiently. We furthermore demonstrate that purification from the endogenous pool using the Strep-tag system results in functionally active complexes. Finally, using the fluorescent tags, we show that a kinase and a phosphatase involved in regulating the activity of the PDH complex localize in the cells’ mitochondria. In conclusion, our cassettes can be used as tools for biochemical, functional, and structural analyses of endogenous multi-protein assemblies in yeast.     

A finite-state continuous-time approach for inferring regional migration and mortality rates from archival tagging and conventional tag-recovery experiments

Spatially structured population dynamics models are important management tools for harvested, highly mobile species and although conventional tag recovery experiments remain useful for estimation of various demographic parameters of these models, archival tagging experiments are becoming an important data source for analyzing migratory behavior of mobile marine species. We provide a likelihood-based approach for estimating the regional migration and mortality rate parameters intrinsic to these models that may use information obtained from conventional tag recovery and archival tagging experiments. Specifically, we assume that the regional location and survival of animals through time is a finite-state continuous-time stochastic process. The stochastic process is the basis of probability models for observations provided by the different types of tags. Results from application to simulated tagging experiments for western Atlantic bluefin tuna show that maximum likelihood estimators based on archival tagging observations and corresponding confidence intervals perform similar to conventional tagging observations for a given number of tag releases and releasing tags in each region can improve the behavior of maximum likelihood estimators regardless of tag type. We provide an example application with Atlantic bluefin tuna released with conventional tags in 1990-1992.     

Lipid phase transitions in cat oocytes supplemented with deuterated fatty acids

Cryopreservation of oocytes has already been used to preserve genetic resources, but this technology faces limitations when applied to the species whose oocytes contain large amounts of cytoplasmic lipid droplets. Although cryoinjuries in such oocytes are usually associated with the lipid phase transition in lipid droplets, this phenomenon is still poorly understood. We applied Raman spectroscopy of deuterium-labeled lipids to investigate the freezing of lipid droplets inside cat oocytes. Lipid phase separation was detected in oocytes cryopreserved by slow-freezing protocol. For oocytes supplemented with stearic acid, we found that saturated lipids form the ordered phase being distributed at the periphery of lipid droplets. When an oocyte is warmed to physiological temperatures after cooling, a fraction of saturated lipids may remain in the ordered conformational state. The fractions of monounsaturated and polyunsaturated lipids redistribute to the core of lipid droplets. Monounsaturated lipids undergo the transition to the ordered conformational state below −10°C. Using deuterated fatty acids with a different number of double bonds, we reveal how different lipid fractions are involved in the lipid phase transition of a cytoplasmic lipid droplet and how they can affect cell survival. Raman spectroscopy of deuterated lipids has proven to be a promising tool for studying the lipid phase transitions and lipid redistributions inside single organelles within living cells.     

Lipid-gene regulatory network reveals coregulations of triacylglycerol with phosphatidylinositol/lysophosphatidylinositol and with hexosyl-ceramide

Lipid homeostasis is important for executing normal cellular functions and maintaining physiological conditions. The biophysical properties and intricate metabolic network of lipids underlie the coordinated regulation of different lipid species in lipid homeostasis. To reveal the homeostatic response among different lipids, we systematically knocked down 40 lipid metabolism genes in Drosophila S2 cells by RNAi and profiled the lipidomic changes. Clustering analyses of lipids reveal that many pairs of genes acting in a sequential fashion or sharing the same substrate are tightly clustered. Through a lipid-gene regulatory network analysis, we further found that a reduction of triacylglycerol (TAG) is associated with an increase of phosphatidylinositol (PI) and lysophosphatidylinositol (LPI) or a reduction of hexosyl-ceramide (HexCer) and hydroxylated hexosyl-ceramide (OH-HexCer). Importantly, negative coregulation between TAG and LPI/PI, and positive coregulation between TAG and HexCer, were also found in human Hela cells. Together, our results reveal coregulations of TAG with PI/LPI and with HexCer in lipid homeostasis.     

Visual monitoring of post-translational lipid modifications using EGFP-GTPase probes in live cells

Modification of small GTPases by lipids is required for their proper subcellular localization and biological activity. Lipids added post-translationally include both farnesyl and geranylgeranyl isoprenoids and the fatty acid palmitate. Thus, specific small molecule inhibitors of these processes cause mislocalization of small GTPases and impair their biological activity. Common biochemical methods of determining the lipid modification status or inhibitor sensitivity of small GTPases, such as in vitro prenylation assays, SDS-PAGE mobility shifts or metabolic labeling, although highly useful in their own right, cannot distinguish differences among specific subpopulations of cells, link lipid modification status with other properties of interest, or provide spatio-temporal information. An alternative method takes advantage of the tight link between small GTPase lipid modification and subcellular localization. The innate localization pattern of the enhanced green fluorescent protein, a common epitope tag frequently used in live cell imaging, is altered by fusion to modified but not unmodified small GTPases. We describe here a technique that takes advantage of these properties to monitor post-translational modifications of these proteins in a rapid, visual manner in live cells.     

Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.     

Using cyclodextrin-induced lipid substitution to study membrane lipid and ordered membrane domain (raft) function in cells

Methods for efficient cyclodextrin-induced lipid exchange have been developed in our lab. These make it possible to almost completely replace the lipids in the outer leaflet of artificial membranes or the plasma membranes of living cells with exogenous lipids. Lipid replacement/substitution allows detailed studies of how lipid composition and asymmetry influence the structure and function of membrane domains and membrane proteins. In this review, we both summarize progress on cyclodextrin exchange in cells, mainly by the use of methyl-alpha cyclodextrin to exchange phospholipids and sphingolipids, and discuss the issues to consider when carrying out lipid exchange experiments upon cells. Issues that impact interpretation of lipid exchange are also discussed. This includes how overly naïve interpretation of how lipid exchange-induced changes in domain formation can impact protein function.     

Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes

The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.     

Improved Lower Bounds of DNA Tags Based on a Modified Genetic Algorithm

The well-known massively parallel sequencing method is efficient and it can obtain sequence data from multiple individual samples. In order to ensure that sequencing, replication, and oligonucleotide synthesis errors do not result in tags (or barcodes) that are unrecoverable or confused, the tag sequences should be abundant and sufficiently different. Recently, many design methods have been proposed for correcting errors in data using error-correcting codes. The existing tag sets contain small tag sequences, so we used a modified genetic algorithm to improve the lower bound of the tag sets in this study. Compared with previous research, our algorithm is effective for designing sets of DNA tags. Moreover, the GC content determined by existing methods includes an imprecise range. Thus, we improved the GC content determination method to obtain tag sets that control the GC content in a more precise range. Finally, previous studies have only considered perfect self-complementarity. Thus, we considered the crossover between different tags and introduced an improved constraint into the design of tag sets.     

Lipid mimetics: A versatile toolbox for lipid biology and beyond

Being the principal component of biological membranes lipids are essential building blocks of life. Given their huge biological importance, the investigation of lipids, their properties, interactions and metabolic pathways is of prime importance for the fundamental understanding of living cells and organisms as well as the emergence of diseases. Different strategies have been applied to investigate lipid-mediated biological processes, one of them being the use of lipid mimetics. They structurally resemble their natural counterparts but are equipped with functionality that can be used to probe or manipulate lipid-mediated biological processes and biomembranes. Lipid mimetics therefore constitute an indispensable toolbox for lipid biology and membrane research but also beyond for potential applications in medicine or synthetic biology. Herein, we highlight recent advances in the development and application of lipid-mimicking compounds.