Detection of Mercury in Aquatic Environments Using EPRE Reporter Zebrafish

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase–green fluorescent protein (LUC–GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl₂ concentrations. GFP expression from the EPRE–LUC–GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl₂. We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl₂ concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.     

In vivo and in vitro protein solubility assays using split GFP

The rapid assessment of protein solubility is essential for evaluating expressed proteins and protein variants for use as reagents for downstream studies. Solubility screens based on antibody blots are complex and have limited screening capacity. Protein solubility screens using split beta-galactosidase in vivo and in vitro can perturb protein folding. Split GFP used for monitoring protein interactions folds poorly, and to overcome this limitation, we recently developed a protein-tagging system based on self-complementing split GFP derived from an exceptionally well folded variant of GFP termed 'superfolder GFP'. Here we present the step-by-step procedure of the solubility assay using split GFP. A 15-amino-acid GFP fragment, GFP 11, is fused to a test protein. The GFP 1-10 detector fragment is expressed separately. These fragments associate spontaneously to form fluorescent GFP. The fragments are soluble, and the GFP 11 tag has minimal effect on protein solubility and folding. We describe high-throughput protein solubility screens amenable both for in vivo and in vitro formats. The split-GFP system is composed of two vectors used in the same strain: pTET GFP 11 and pET GFP 1-10 (Fig. 1 and Supplementary Note online). The gene encoding the protein of interest is cloned into the pTET GFP 11 vector (resulting in an N-terminal fusion) and transformed into Escherichia coli BL21 (DE3) cells containing the pET GFP 1-10 plasmid. We also describe how this system can be used for selecting soluble proteins from a library of variants (Box 1). The large screening power of the in vivo assay combined with the high accuracy of the in vitro assay point to the efficiency of this two-step split-GFP tool for identifying soluble clones suitable for purification and downstream applications.     

Shotgun Crystallization Strategy for Structural Genomics II: Crystallization Conditions that Produce High Resolution Structures for T. maritima Proteins

Currently, 119 high resolution structures of Thermotoga maritima proteins have been determined by the Joint Center for Structural Genomics (JCSG, www.jcsg.org). Sixty-seven of these were solved using the first implementation of the multi-tiered crystallization strategy at the JCSG for the efficient crystallization of large numbers of protein targets. Previously, we reported the analysis of all proteins crystallized using this multi-tiered strategy [Lesley, S.A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 11664–11669; Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037]. Here, we extend the analysis and describe the crystallization characteristics of those proteins that produced diffraction quality crystals, ultimately resulting in high resolution structures. First, we found that over 77% (52) of the crystals used for structure determination were produced directly from high-throughput coarse screens, indicating that less than one quarter of the crystals (15) required fine screening. In addition, as observed for the proteome screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037], the majority of conditions that produced crystals for natively expressed proteins, whose structures have been determined, were distinct from those of their more extensively purified and selenomethionine-labeled counterparts. Finally, 99% of the proteins whose structures were solved crystallized in conditions contained in the JCSG Minimal Core Screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037; Page, R. and Stevens, R.C. (2004) Methods 34, 373–389], a set of 67 conditions previously identified as those most likely to produce crystals of a diverse set of proteins, confirming its success for rapid identification of proteins with a natural propensity to crystallize.     

An improved method for large-scale purification of recombinant human glucagon

Glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence [Ishizakiet al. (1992),Appl. Microbiol. Biotechnol.36, 483–486]. The high-level expression of a protein inE. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawaet al. (1992),J. Protein Chem. 11, 517–525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon.     

Folding of green fluorescent protein and the cycle3 mutant

Although the correct folding of green fluorescent protein (GFP) is required for formation of the chromophore, it is known that wild-type GFP cannot mature efficiently in vivo in Escherichia coli at 37 degrees C or higher temperatures that the jellyfish in the Pacific Northwest have never experienced. Recently, by random mutagenesis by the polymerase chain reaction (PCR) method, a mutant called Cycle3 was constructed. This mutant had three mutations, F99S, M153T, and V163A, on or near the surface of the GFP molecule and was able to mature correctly even at 37 degrees C [Crameri et al. (1996) Nat. Biotechnol. 143, 315-319]. In the present study, we investigated the differences in their folding behavior in vitro. We observed the folding and unfolding reactions of both wild-type GFP and the Cycle3 mutant by using green fluorescence as an indicator of the formation of the native structure and examining hydrogen-exchange reactions by Fourier transform infrared spectroscopy. Both proteins showed unusually slow refolding and unfolding rates, and their refolding rates were almost identical under the native state at 25 and at 35 degrees C. On the other hand, aggregation studies in vitro showed that wild-type GFP had a strong tendency to aggregate, while the Cycle3 mutant did not. These results indicated that the ability to mature efficiently in vivo at 37 degrees C was not due to the improved folding and that reduced hydrophobicity on the surface of the Cycle3 mutant was a more critical factor for efficient maturation in vivo.     

New molecular reporters for rapid protein folding assays

The GFP folding reporter assay uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility, but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter and the robustly-folding "superfolder" GFP. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37 degrees C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites.     

Versatile signal peptide of Flavobacterium‐originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane‐associated homodimeric metalloenzyme and has its own signal peptide in its N‐terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide‐containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin‐arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec‐avoidance sequence in the c‐region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848–854, 2016     

Green fluorescent proteins with short half-lives as reporters in Dictyostelium discoideum

 We describe two modifications of the popular reporter green fluorescent protein (GFP) which have short half-lives in our system, the cellular slime mould Dictyostelium discoideum. One of these bears an N-terminal ubiquitin; this GFP was originally planned to be a substrate of the ”N-end-rule” pathway, but deubiquitination does not seem to occur, and a degradation by the UFD (ubiquitin-fusion-degradation pathway seems more probable. The protein half-life is about 3–5 h. The second construct has an N-terminus derived from the L11 ribosomal protein; it is transported to the nucleus and broken down much more rapidly than the ubiquitin fusion (protein half-life about 30 min). We show examples of the use of these reporters in the study of gene expression in Dictyostelium. Received: 20 April 1998 / Accepted: 23 August 1998     

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014. © 2014 Wiley Periodicals, Inc.     

Evolution of an Arbitrary Sequence in Solubility

We have investigated the evolvability of an insoluble random polypeptide, RP3-34, to a soluble form through iterative mutation and selection with the aid of the green fluorescent protein (GFP) folding reporter. To assess the solubility of the polypeptides in the selected clones of each generation, the polypeptide genes were detached from the GFP fusions and expressed with a His₆ tag. The solubility of the variant random polypeptides increased in each generation within the scope of the evolutionary process, and the polypeptides assumed a soluble form from the fourth generation. Analysis of the synonymous and nonsynonymous mutations found in the deduced amino acid sequence of the selected polypeptides revealed that selection had accelerated the evolutionary rate. The solubility and hydrophobicity of the polypeptides and the 25 arbitrarily chosen random polypeptides found in a previously prepared library were determined, analyzed, and interpreted from the landscape on the protein sequence space. This study showed the evolvability of an insoluble arbitrary sequence toward a soluble one, hence, it provides a new perspective on the field of artificial evolution.     

Back to Units of Protein Folding

Abstract

In response to the criticism by A. Finkelstein (J. Biomol. Struct. Dyn. 20, 311–314, 2002) of our Communication (J. Biomol. Struct. Dyn. 20, 5–6, 2002) several issues are dealt with. Importance of the notion of elementary folding unit, its size and structure, and the necessity of further characterization of the units for the elucidation of the protein folding in vivo are discussed. The criticism (J. Biomol. Struct. Dyn. 20, 311–314, 2002) on the hierarchical protein folding is also briefly addressed.     

Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish (Danio rerio) Tissues

We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-μm microcarriers and 1 μg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 μg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described. Received August 20, 1999; accepted January 6, 2000.     

Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the secretory production of heterologous proteins via the Sec system. In this study, the potential for Tat-dependent heterologous protein secretion was compared in the three Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis, and Corynebacterium glutamicum using green fluorescent protein (GFP) as a model protein. In all three microorganisms, fusion of a Tat signal peptide to GFP resulted in its Tat-dependent translocation across the corresponding cytoplasmic membranes. However, striking differences with respect to the final localization and folding status of the exported GFP were observed. In S. carnosus, GFP was trapped entirely in the cell wall and not released into the supernatant. In B. subtilis, GFP was secreted into the supernatant, however, in an inactive form. In contrast, C. glutamicum effectively secreted active GFP. Our results clearly demonstrate that a comparative evaluation of different Gram-positive host microorganisms is a crucial step on the way to an efficient Tat-mediated secretory production process for a desired heterologous target protein. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This paper is dedicated to Hermann Sahm on the occasion of his 65th birthday.     

Reporter system for the detection of in vivo gene conversion

We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr⁶⁶-His⁶⁶). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reported cells using a variety of methodologies and strategies. Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.     

Bifunctional Reporter Genes: Construction and Expression in Prokaryotic and Eukaryotic Cells

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli -glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65°C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C or N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.     

Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1α enhancer-promoter (EF1). Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system, were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression in living cells is useful for characterizing the activity of cis-elements in vivo. Received December 21, 1998; accepted March 31, 1999.     

表达GFP/mCherry白色念珠菌的构建及其在与巨噬细胞互作中的应用

白色念珠菌(Candida albicans)被巨噬细胞吞噬的效率与被吞噬后的形态观察是研究白色念珠菌与巨噬细胞互作的重要内容。【目的】以野生型菌株SC5314为母本,构建能够表达绿色荧光蛋白(green fluorescent protein, GFP)/mCherry的白色念珠菌,应用于巨噬细胞与白色念珠菌互作的研究。【方法】通过生长与形态观察、细胞活性检测及小鼠系统性感染模型确定荧光蛋白的表达对菌株生长、形态与毒力的影响;在共培养条件下,通过流式细胞术及荧光显微镜检测巨噬细胞的吞噬率及白色念珠菌的形态变化。【结果】构建的菌株在表型上与野生型菌株一致,并可用于在共培养下测定巨噬细胞吞噬率的流式细胞术以及观察白色念珠菌的形态变化。【结论】表达荧光蛋白的菌株为研究巨噬细胞与白色念珠菌的互作提供了新方法。     

Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans , and its use as a reporter of gene regulation

Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicansACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the non-canonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans. Received: 27 June 1997 / Accepted: 26 September 1997     

Comparative analysis of the protein folding activities of two chaperonin subunits of Thermococcus strain KS-1: the effects of beryllium fluoride

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the α- and β-subunit homooligomers (α16mer or β16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both α16mer and β16mer with equal efficiency. This indicated that BeFx replaces the γ-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin–ADP–BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin–ADP–BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of α16mer and β16mer are likely similar although there were some apparent differences. While β16mer-mediated GFP refolding in the presence of ATP–BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP–PNP, the folding mediated by the α16mer that proceeded with much lower yields. A mutant of α16mer, trapα, which traps the unfolded and partially folded substrate protein, did not affect the ATP–BeFx-dependent GFP folding by β16mer but it suppressed that mediated by α16mer to the level of spontaneous folding. These results suggested that β16mer differed from the α16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis.     

Utilization of Methyl Proton Resonances in Cross-Saturation Measurement for Determining the Interfaces of Large Protein–Protein Complexes

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220–223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of ¹H₂O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.