Identification of GFP-like Proteins in Nonbioluminescent,Azooxanthellate Anthozoa Opens New Perspectives for Bioprospecting

We screened nonbioluminescent, azooxanthellate cnidaria as potential sources for advanced marker proteins and succeeded in cloning a tetrameric green fluorescent protein (GFP) from the tentacles of Cerianthus membranaceus. The fluorescence of this protein (cmFP512) is characterized by excitation maximum at 503 nm, emission maximum at 512 nm, extinction coefficient of 58,800 M^–1 cm^–1, quantum yield of 0.66, and fluorescence lifetime of 2.4 ns. The chromophore is formed from the tripeptide Gln-Tyr-Gly. The amino acid sequence of this protein shares 17.8% identical residues with GFP from Aequorea victoria. Weak interactions between the subunits of the tetramer make cmFP512 a promising lead structure for the generation of monomeric variants of fluorescent proteins. Both red fluorescent proteins and nonfluorescent proteins of the GFP family were also purified from tissue homogenates of Adamsia palliata and Calliactis parasitica. The results presented here indicate that a photoprotective function of GFP-like proteins is unlikely in the examined anthozoa species.     

Characterization of Novel Orange Fluorescent Protein Cloned from Cnidarian Tube Anemone <Emphasis Type="Italic">Cerianthus</Emphasis> sp.

A novel orange fluorescent protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. It consists of 222 amino acid residues with a calculated molecular mass of 25.1 kDa. A BLAST protein sequence homology search revealed that native OFP has 81% sequence identity to Cerianthus membranaceus green fluorescent protein (cmFP512), 38% identity to Entacmaea quadricolor red fluorescent protein (eqFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-orange fluorescent protein (KO), and a mere 21% identity to green fluorescent protein (GFP). It is most likely that OFP also adopts the 11-strand β-barrel structure of fluorescent proteins. Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 487 and 513 nm. A bright orange fluorescence maximum at 573 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 515 nm, a considerable amount of green emission maximum at 513 nm was also observed. It has a fluorescence quantum yield (Φ) of 0.64 at 25°C. The molar absorption coefficients (ɛ) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M-1⁻¹ • cm-1⁻¹, respectively. Its fluorescent brightness (ɛ Φ) at 25°C is 38,400 M⁻¹-1 • cm⁻¹-1. Like other orange-red fluorescent proteins, OFP is also tetrameric. It was readily expressed as soluble protein in Escherichia coli at 37°C, and no aggregate was observed in transfected HeLa cells under our experimental conditions. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12.     

A Synthesis of New Rigid Fluorescent Bichromophoric Probes for Studying Mechanisms of Donor-Donor Energy Migration

Three new fluorescent probes were synthesized for improving the method of studying donor-donor energy migration (DDEM). Each probe has two identical fluorescent 7-diethylaminocoumarin-3-carbonyl groups attached to a rigid bisteroid dodecacyclic spacer through additional inserts. In two probes, the inserts are β-Ala and L-Ser residues, which provide for a different nearest environment of the fluorophores. The third probe has identical β-Ala inserts.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 331–334.Original Russian Text Copyright © 2005 by Boldyrev, Molotkovsky.     

Characteristics of natural strains of naphthalene-utilizing bacteria of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Sixty-three strains of bacteria capable of utilizing naphthalene as the sole source of carbon and energy were isolated from 137 samples of soil taken in different sites in Belarus. All isolated bacteria contained extrachromosomal genetic elements of 45 to 150 kb in length. It was found that bacteria of 31 strains contained the IncP-9 incompatibility group plasmids, bacteria of one strain carried a plasmid containing replicons IncP-9 and IncP-7, and bacteria of 31 strains contained unidentified plasmids. Primary identification showed that the hosts of plasmids of naphthalene biodegradation are fluorescent bacteria of the genus Pseudomonas (P. putida and P. aeruginosa; a total of 47 strains) and unidentified nonfluorescent microorganisms (a total of 16 strains). In addition to the ability to utilize naphthalene, some strains exhibited the ability to stimulate the growth and development of the root system of Secale cereale.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 162–167.Original Russian Text Copyright © 2005 by Levchuk, Vasilenko, Bulyga, Titok, Thomas.     

A Natural Fluorescent Protein That Changes Its Fluorescence Color during Maturation

The gene of a new red fluorescent protein zoan2RFP from coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the jellyfish Aequorea victoria, was cloned. At early stages of maturation, zoan2RFP exhibits green fluorescence, which then turns to the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.     

A method for the preparation of normalized cDNA libraries enriched with full-length sequences

We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, effectively cleaves double-stranded DNA, and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935–1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 186–194.Original Russian Text Copyright © 2005 by Zhulidov, Bogdanova, Shcheglov, Shagina, Wagner, Khazpekov, Kozhemyako, Lukyanov, Shagin.     

A tunable fluorescent timer method for imaging spatial‐temporal protein dynamics using light‐driven photoconvertible protein

Cellular function is largely determined by protein behaviors occurring in both space and time. While regular fluorescent proteins can only report spatial locations of the target inside cells, fluorescent timers have emerged as an invaluable tool for revealing coupled spatial‐temporal protein dynamics. Existing fluorescent timers are all based on chemical maturation. Herein we propose a light‐driven timer concept that could report relative protein ages at specific sub‐cellular locations, by weakly but chronically illuminating photoconvertible fluorescent proteins inside cells. This new method exploits light, instead of oxygen, as the driving force. Therefore its timing speed is optically tunable by adjusting the photoconverting laser intensity. We characterized this light‐driven timer method both in vitro and in vivo and applied it to image spatiotemporal distributions of several proteins with different lifetimes. This novel timer method thus offers a flexible “ruler” for studying temporal hierarchy of spatially ordered processes with exquisite spatial‐temporal resolution. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

灰葡萄孢菌过氧化物酶体及细胞核的荧光蛋白标记

【目的】对灰葡萄孢菌(Botrytis cinerea)的细胞核和过氧化物酶体进行荧光蛋白标记,为研究其生长发育和侵染过程中细胞结构和细胞器动态提供基础。【方法】以绿色荧光蛋白(GFP)和红色荧光蛋白(DsRED、mCherry)为报告基因,利用根癌农杆菌介导转化(Agrobacterium tumefaciens mediated transformation,AtMT)将3种荧光蛋白标记载体分别导入灰葡萄孢菌标准菌株B05.10;通过PCR检测及荧光观察筛选和验证转化子,并进行单孢纯化;利用共聚焦显微镜记录细胞器荧光定位情况。【结果】获得了过氧化物酶体或细胞核稳定表达红、绿色荧光的重组单孢菌株,PCR验证表明标记基因成功整合入转化子基因组。在标记细胞核的菌株中,菌丝和孢子中可见多个明亮、圆形的荧光点,与DAPI染色共定位。标记过氧化物酶体的菌株中,菌丝和孢子中可见小点状绿色或红色荧光,在脂类物质诱导下荧光点数量明显增加,符合过氧化物酶体分布及动态特征。细胞壁染色结果显示,细胞壁染色产生的蓝色荧光与红、绿荧光蛋白的荧光互不干扰,标记效果良好。【结论】获得了理想的过氧化物酶体或细胞核荧光标记的灰葡萄孢菌菌株,为研究其细胞器动态以及生长发育与致病分子机制提供了参考和材料。     

Probing the structural determinants of yellow fluorescence of a protein from Phialidium sp

Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λ_em = 537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling – mass spectrometry approach. Mass spectrometry of the isolated chromophore-bearing peptide reveals that the chromophore of phiYFP is chemically identical to that of avGFP (λ_em = 508 nm). The experimentally acquired chromophore structure was combined with the homology-based model of phiYFP, and the proposed 3D structure was used as a starting point for identification of the structural features responsible for yellow fluorescence. Mutagenesis of residues in the local chromophore environment of phiYFP suggests that multiple factors cooperate to establish the longest-wavelength emission maximum among fluorescent proteins with an unmodified GFP-like chromophore.     

Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21–KlMAL22 bi-directional promoter

We have identified the Kluyveromyces lactis maltase (KlMAL22) and maltose permease (KlMAL21) intergenic region as a candidate bi-directional promoter for heterologous gene expression. The expressions of cyan and yellow fluorescent proteins from, respectively, the KlMAL22 and KlMAL21 orientations of the promoter, were compared between two promoter variants during growth in media containing glucose, galactose or glycerol. Expression from both orientations of the native promoter was repressed during growth in glucose and galactose and was induced during growth in glycerol. Disruption of a putative Mig1p binding site caused some de-repression of the maltase orientation of the promoter by 48 h of growth in glucose. The KlMAL21–-KlMAL22 bi-directional promoter can be used to carry out regulated expression of heterologous gene products.     

Binding of chimeric metal-binding green fluorescent protein to lipid monolayer

Membrane-based bioanalytical devices for metal determination using green fluorescent protein as the sensor molecule may be a useful future biomimetic material. However, in order to develop such a device, it is necessary first to understand the interaction of the protein with lipid membranes. Thus we have investigated the interaction between chimeric cadmium-binding green fluorescent proteins (CdBPGFPs) and lipid monolayers, using a film-balance technique complemented with epifluorescence microscopy. The binding avidity was monitored from the surface pressure vs. area isotherms or from the measured increase in the lateral pressure upon injection of the chimeric CdBPGFPs beneath the lipid monolayer. Increased fluidization as well as expansion of the surface area were shown to depend on the concentration of the CdBPGFPs. The kinetics of the protein-induced increase in lateral pressure was found to be biphasic. The chimeric CdBPGFPs possessed high affinity to the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer with a dissociation constant of K_d=10^–8M. Epifluorescence measurements showed that this affinity is due to the presence of the Cd-binding peptide, which caused the GFP to incorporate preferentially to the liquid phase and defect part of the rigid domain at low interfacial pressure. At high compression, the Cd-binding peptide could neither incorporate nor remain in the lipid core. However, specific orientation of the chimeric CdBPGFPs underneath the air–water interface was achieved, even under high surface pressure, when the proteins were applied to the metal-chelating lipid-containing surfaces. This specific binding could be controlled reversibly by the addition of metal ions or metal chelator. The reversible binding of the chimeric CdBPGFPs to metal-chelating lipids provided a potential approach for immobilization, orientation and lateral organization of a protein at the membrane interface. Furthermore, the feasibility of applying the chelator lipids for the codetermination of metal ions with specific ligands was also revealed. Our finding clearly demonstrates that a strong interaction, particularly with fluid lipid domains, could potentially be used for sensor development in the future.Abbreviations GFP green fluorescent protein - CdBPGFPs cadmium-binding green fluorescent protein - DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - AAS atomic absorption spectrometry - Cd²⁺ cadmium (II) - Zn²⁺ zinc (II) - Cu²⁺ copper (II) - Ni²⁺ nickel (II) - E. coli Escherichia coli - NTA-DOGS 1,2-dioleoyl-sn-glycero-3-(N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl) - His6GFP hexahistidine green fluorescent protein - CdBP4GFP four-repeat cadmium-binding peptide green fluorescent protein - His6CdBP4GFP hexahistidine four-repeat cadmium-binding peptide green fluorescent protein - IMAC immobilized-metal-affinity chromatography - PBS phosphate-buffered saline - mN/m millinewton per metre - le liquid expanded - lc liquid condensed - PE phosphatidyl ethanolamine - PI phosphatidyl inositol - NTA nitrilotriacetic acid - EDTA ethylenediamine tetraacetic acid - RESA ring-infected erythrocyte surface antigen - CdBP cadmium-binding peptide     

Fluorescent protein biosensors applied to microphysiological systems

This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS.     

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     

Creation of Circularly Permutated Yellow Fluorescent Proteins Using Fluorescence Screening and a Tandem Fusion Template

By experimenting with many different circularly permutated yellow fluorescent protein (cpYFP) variants as acceptors in fluorescence resonance energy transfer based biosensors, the optimal dynamic range can be discovered by sampling the possibilities of relative fluorophore orientations before and after bioactivity. Hence, to facilitate the sampling process, we introduced a new approach to construct a library of cpYFP variants using fluorescence screening and a tandem fusion template. This new approach is rapid because it does not require creating intermediate N- and C-terminal fragments and it allows quick screening for positive colonies by fluorescence. As a demonstration, eleven cpYFP variants were created and eight showed fluorescence. The emission and excitation spectra of these cpYFP variants showed strong similarity to YFP and therefore can be used in replacement. Revisions requested 27 October 2005; Revisions received 23 December 2005     

Fluorescent method for studying the morphogenesis and viability of dermatophyte cells

The dermatophyte Microsporum canis is commonly isolated from human and animal infection. The morphogenesis of this fungus was studied during its developmental stages through the fluorescent method Fluorescein Diacetate and Ethidium Bromide. To this end, 50 l dermatophyte suspension were transferred onto cellophane wrapping esterilized discs (2.5 cm of diameter) placed over the surface of Sabouraud dextrose agar on Petri dishes and incubated at 25 °C for 30 days. Every 60 minutes during the first 24 hours and every 12 hours for next 29 days, one disc was transferred onto glass slide, covered with equal volumes of freshly prepared fluorescein diacetate (FDA) and ethidium bromide (EB) solution, mounted with a coverslip and incubated in the dark for 30 minutes, at 25 °C. Each preparation was then examined on a fluorescent microscope. M. canis presented well defined growth stages: (1) tumescence of cells; (2) germination; (3) development of hyphae; (4) production of conidia and (5) tumescence and formation of arthroconidiae. Using the fluorescent method, non viable cells showed a light bright red coloration and viable cells presented green fluorescence. The principal morphological changes have occurred between the 3rd until the 18th day of culture. The method is very useful to demonstrate the dermatophyte growth stages as well as the perfect differentiation between viable and non viable cells.This revised version was published online in October 2005 with corrections to the Cover Date.     

DEVELOPMENT OF GENETICALLY ENCODED FLUORESCENT PROTEIN CONSTRUCTS OF HYPERTHERMOPHILIC MALTOSE-BINDING PROTEIN

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.     

红色荧光蛋白的光谱多样性及体外分子进化

从珊瑚中来源的各种红色荧光蛋白（red fluorescent protein,RFP）经过一系列体外进化,其波谱范围覆盖了570－655mn,极大地丰富了细胞内或体内光学成像的荧光探针．简要阐述了来源于Discosoma sp．,Entacmaea quadricolor,Anemonia sulcata,Heteractis crispo,Actinia equina5种珊瑚的红色荧光蛋白的光学特征、结构、体外分子进化及其应用．     

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large‐scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two‐phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle‐poor phase (K_GFPuv < 1.00), and LPS removal into the micelle‐rich phase (%REM_LPS > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High‐yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii

Microalga‐based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post‐translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C‐terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)_n, wherein n = 10 or 20]. The yields of the (SP)_n‐fused Venus were higher than Venus without the glycomodule by up to 12‐fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus‐(SP)_n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)_n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)_n glycomodule to promote a more efficient biomanufacturing of microalgae‐based recombinant proteins.     

Near-infrared, dual-ratiometric fluorescent label for measurement of pH

We describe the spectral properties of an amine-reactive, pH-sensitive, long-wavelength ratiometric fluorescent label having a pK_a in the physiological pH range. The label exhibits its main absorption and emission in the near-infrared (NIR) region. On deprotonation, a blue shift of the excitation maximum is observed. Importantly, both the protonated and deprotonated forms of the label are fluorescent, with the deprotonated form having an extremely large Stokes shift of more than 100 nm. The spectral and photophysical properties of this pH label are compared with the properties of the protein-conjugated forms. Due to the observed pK_a shift to the acidic pH range upon conjugation to proteins, such labels are ideal for studying phagocytic events and their regulation by drugs and/or environmental factors.