Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution

We previously reported a method, designated functional salvage screen (FSS), to generate protein lineages with new sequence spaces through the functional or structural salvage of a defective protein by employing green fluorescent protein (GFP) as a model protein. Here, in an attempt to mimic a step in the natural evolution process of proteins, the functionally salvaged mutant GFP-I5 with new sequence space, but showing low fluorescence intensity and stability, was selected and fine-tuned by directed evolution. During a course of functional tuning, GFP-I5 was found to evolve rapidly, recovering the spectral traits to those of the parent GFPuv. The mutant 3E4 from the third round of directed evolution possessed four substitutions; three (F64L, E111V and K166Q) were at the original GFP gene and the other (K8N) at the inserted segment. The fluorescence intensity of 3E4 was approximately 28-fold stronger than GFP-I5, and other spectral properties were retained. Biochemical and biophysical investigations suggested that the fine-tuning by directed evolution led the salvaged variant GFP-I5 to a functionally favorable structure, resulting in recovery of stability and fluorescence. Site-directed mutagenesis of the mutated amino acid residues in both GFPuv and GFP-I5 revealed that each amino acid residue has a different effect on the fluorescence intensity, which implies that 3E4 adopted a new evolutionary path with respect to fluorescence characteristics compared with the parent GFPuv. Directed evolution in conjunction with FSS is expected to be used for generating protein lineages with new fitness landscapes.     

Protein Evolution via Amino Acid and Codon Elimination

Background

Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.

Methodology/Principal Findings

The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.

Conclusions/Significance

The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.     

Deletional Protein Engineering Based on Stable Fold

Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.     

A potyvirus vector efficiently targets recombinant proteins to chloroplasts,mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein

Plant virus‐based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino‐terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino‐terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus‐based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering.     

Metal-enhanced fluorescence of single green fluorescent protein (GFP)

The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer duration time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.     

Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression

Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.     

In vitro stability and expression of green fluorescent protein under high pressure conditions

AIMS: The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions. METHODS AND RESULTS: The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All tested GFP's retained fluorescence up to 600 MPa without loss of intensity. Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site. T7 RNA polymerase is controlled in E. coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa. CONCLUSION: Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs. We showed that the expression system used is inducible by pressurized conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression.     

Engineering green fluorescent protein as a dual functional tag

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

N-Terminal Protein Acylation Confers Localization to Cholesterol, Sphingolipid-enriched Membranes But Not to Lipid Rafts/Caveolae

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.     

Characterization and Use of Green Fluorescent Proteins from Renilla mulleri and Ptilosarcus guernyi for the Human Cell Display of Functional Peptides

Green fluorescent protein (GFP) is useful as an intracellular scaffold for the display of random peptide libraries in yeast. GFPs with a different sequence from Aequorea victoria have recently been identified from Renilla mulleri and Ptilosarcus gurneyi. To examine these proteins as intracellular scaffolds for peptide display in human cells, we have determined the expression level of retrovirally delivered human codon-optimized versions in Jurkat-E acute lymphoblastic leukemia cells using fluorescence activated cell sorting and Western blots. Each wild type protein is expressed at 40% higher levels than A. victoria mutants optimized for maximum fluorescence. We have compared the secondary structure and stability of these GFPs with A. victoria GFP using circular dichroism (CD). All three GFPs essentially showed a perfect -strand conformation and their melting temperatures (T_m) are very similar, giving an experimental evidence of a similar overall structure. Folded Renilla GFP allows display of an influenza hemagglutinin epitope tag in several internal insertion sites, including one which is not permissive for such display in Aequorea GFP, giving greater flexibility in peptide display options. To test display of a functional peptide, we show that the SV-40 derived nuclear localization sequence PPKKKRKV, when inserted into two different potential loops, results in the complete localization of Renilla GFP to the nucleus of human A549 cells.     

A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins

Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein.     

Stubborn GFPs in Nicotiana tabacum vacuoles

Green fluorescent protein (GFP) allows the direct visualization of gene expression and sub cellular localization of fusion proteins in living cells. Many GFP variants have been developed to solve stability and emission problems. In this report the localization of different GFP fusion proteins, targeted to vacuoles, was studied in Nicotiana tabacum cv SR1. Even if a strong emission variant of the plant adapted GFP was used, no fluorescence was detected in differentiated tissues of N. tabacum with few exceptions. This model plant does not appear a good experimental system for the use of GFPs as vacuolar markers compared to Arabidopsis thaliana. In spite of this, our observations have evidenced a peculiar pattern of separated vacuoles in guard cells, providing new elements in the understanding of the vacuolar system organization.     

Imaging FRET between spectrally similar GFP molecules in single cells

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.     

Is green fluorescent protein toxic to the living cells?

Green fluorescent protein (GFP) has become more popular to be used as a living marker for positively transfected clones in many studies. To establish stable cell lines constitutively expressing GFP, three GFPs expressed from plasmid pBIEGFP, pSG5GFP, and pRSGFP were introduced into NIH/3T3, BHK-21, Huh-7, and HepG2 cells. All the GFPs we used are the mutant forms of a common wild phenotype. The pBIEGFP expressed enhanced GFP (EGFP). The pRSGFP and pSG5GFP expressed red shift GFP (RSGFP). The RSGFP gene in pSG5GFP was driven by a strong SV40 promoter and showed at least 20-fold higher RSGFP expression by western blot analysis. Despite of the variation in the levels of GFP expression, many GFP expressing cells contracted, rounded-up, and died, which was confirmed by decreasing luciferase activity. CPP32 activity and flow cytometric analyses further demonstrate that cells expressing GFP underwent apoptosis. Our observation is contradictory to other reports that GFP is nontoxic to the cells. Most importantly, this paper shows for the first time the link between expression of GFP and induction of apoptosis. This finding should promote studies of GFP cytotoxicity and attempts to isolate new non-toxic mutants of GFP.     

GFPs of insertion mutation generated by molecular size-altering block shuffling

Insertion and deletion analyses of a protein have been less common than point mutation analyses, partly due to the lack in effective methods. This is the case with the green fluorescent protein (GFP), which is so widely applied in molecular biology and other fields. In this paper we first introduce a systematic approach for generating insertion/deletion mutants of GFP. A new technology of Y-ligation-based block shuffling (YLBS) was successfully applied to produce size-altered GFPs, providing insertion-containing GFPs of fluorescence, though no deletion type of fluorescence was obtained so far as examined. The analysis of these proteins suggested that size alteration (deletion/insertion) is acceptable so far as some type of rearrangement in a local structure can accommodate it. This paper demonstrates that YLBS can generate insertion and deletion mutant libraries systematically, which are beneficial in the study of structure-function relationship.     

Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system

In order to develop a high-level expression system in transgenic rice, we inserted a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (GFP) into two expression vectors, Act1-sgfp for an untargeted and rbcS-Tp-sgfp for a chloroplast targeted expression. Several fertile transgenic rice plants were produced by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that, in cells expressing the Act1-sgfp, GFP fluorescence was localized within the cytoplasm and nucleoplasm whereas, in cells expressing the rbcS-Tp-sgfp fusion gene, the fluorescence was specifically targeted to chloroplasts and non-green plastids. The levels of sgfp expression were about 0.5% of the total soluble protein in mature leaf tissues of the Act1-sgfp transformed lines. In contrast, expression levels were markedly increased in mature leaf tissues of the rbcS-Tp-sgfp transformed lines, yielding about 10% of the total soluble protein. N-terminal sequencing of the localized GFPs revealed that the Tp-GFP fusion protein was correctly processed during import to non-green plastids, as well as to chloroplasts. Thus, our results demonstrate that GFP can be produced at high levels and localized in specific subcellular spaces of transgenic plants, providing a high-level expression system for general use in rice, an agronomically important cereal.     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

Circularly permuted variants of the green fluorescent protein.

Folding of the green fluorescent protein (GFP) from Aequorea victoria is characterized by autocatalytic formation of its p-hydroxybenzylideneimidazolidone chromophore, which is located in the center of an 11-stranded beta-barrel. We have analyzed the in vivo folding of 20 circularly permuted variants of GFP and find a relatively low tolerance towards disruption of the polypeptide chain by introduction of new termini. All permuted variants with termini in strands of the beta-barrel and about half of the variants with termini in loops lost the ability to form the chromophore. The thermal stability of the permuted GFPs with intact chromophore is very similar to that of the wild-type, indicating that chromophore-side chain interactions strongly contribute to the extraordinary stability of GFP.