Secretion Of Green Fluorescent Protein Research Articles

A Model for Pharmaceutical Proteins Production from Arabidopsis Suspension Culture

Pharmaceutical proteins production in plant suspension culture has been of interest due to the ease of manipulation, low cost and low allergenic reaction in humans. In this work, the constitutive double CaMV 35S promoter and the abiotic stress-responsive promoter of the Rd29a locus were compared to produce Green Fluorescent Protein (GFP) used as a model for secreted protein in Arabidopsis thaliana suspension culture. Induction of Rd29a inducible promoter by plant hormone abscisic acid (ABA) was also studied. A signal sequence (SS) of a 33 kDa rice protein was added to the constructs to promote secretion. The secretion of GFP into the extracellular spaces of the transgenic seedling was detected under confocal microscope. The fl uorescence intensity of GFP in the suspension culture was measured by fl uorometry. A temporal maximum expression of GFP was observed from the CaMV 35S promoter on day 18. This model can be used as an example to produce pharmaceutical proteins in suspension culture in the future.

Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system

The power of the CRISPR/Cas9 system has revolutionized genome editing in many fields of biology. These applications have expanded exponentially over recent years, including those regarding protein expression technologies. The CRISPR/Cas9 system avoids random integration of the gene of interest and due to this characteristic can be exploited to obtain a stable cell line for the high-yield expression of recombinant proteins. Here we propose a method to edit a hybridoma cell line for the constitutive expression of proteins of interest using the CRISPR/Cas9 system. First, with the scope of optimizing the method, we replaced part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, obtaining a precise knock-in in the hybridoma genome. We confirmed the expression and secretion of GFP into the culture medium via fluorimetric analysis, as well as correct genome editing by RNA sequencing. Then, using the same approach, we included the gene encoding a protein of diagnostic interest, the Bovine Herpesvirus 1 glycoprotein E, in the donor DNA. We obtained a stable clone able to secrete gE protein in fusion with GFP into the culture medium. This result was confirmed by ELISA and Western Blot analysis. This study confirms the suitability of this cell line for the production of proteins of diagnostic interest by stable gene expression in a mammalian system. These experiments will enable the technique to be developed from its proof of concept to more specific applications in the field of infectious disease diagnostics.

Identification and characterisation of the novel endogenous promoter HASP1 and its signal peptide from Phaeodactylum tricornutum

Although diatoms have been extensively studied as bioreactors, only a limited number of efficient gene promoters are available. Therefore, the development of new endogenous promoters is important for the heterologous production of a variety of recombinant proteins. Herein, we identified the most abundant secreted protein in Phaeodactylum tricornutum, designated ‘highly abundant secreted protein 1’ (HASP1), and characterised the activities of its promoter and signal peptide using green fluorescent protein (GFP) as a reporter. The HASP1 promoter strongly drove GFP expression during all growth phases of P. tricornutum in culture, in contrast to the commonly used fcpA promoter, which is less active during the stationary phase. The HASP1 signal peptide was also sufficient for facilitating efficient secretion of GFP by P. tricornutum. Our findings suggest that both the promoter and the signal peptide of HASP1 can be utilized as novel tools for the overexpression and secretion of recombinant proteins in P. tricornutum.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%–92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

The effect of combining signal sequences with the N28 fragment on GFP production in Aspergillus oryzae

Effective secretion of green fluorescent protein (GFP) was investigated by the screening signal sequences for GFP secretion in Aspergillus oryzae. GFP production in A. oryzae was evaluated using fusions with signal sequences from Taka-amylase A (TAA), glucoamylase A, glucoamylase B, and triacylglycerol lipase. The TAA signal sequence promoted the highest protein secretion of GFP. Fusing this signal sequence with an N-terminal 28-amino acid region (N28 fragment) from the Rhizopus oryzae lipase signal sequence increased protein secretion. In addition, using multiple copies of this signal sequence, instead of the N28 fragment, also induced protein secretion. These results show that using multiple signal sequences or combining a signal sequence with the N28 fragment can be used to improve heterogeneous protein secretion in A. oryzae.

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.

High-Salinity Growth Conditions Promote Tat-Independent Secretion of Tat Substrates in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.

Adeno-Associated Virus Vector-Mediated Expression and Constitutive Secretion of Galanin Suppresses Limbic Seizure Activity

Theoretically, gene therapy techniques offer an attractive alternative treatment option for intractable, focal epilepsies. Although logical gene therapy targets include excitatory and inhibitory receptors, variable viral vector tropism interjects an uncertainty as to the direction of change, seizure suppression, or seizure sensitization. To circumvent this therapeutic liability, adeno-associated virus (AAV) vectors have been constructed where the gene product is constitutively secreted from the transduced cell. Using AAV vectors, the fibronectin secretory signal sequence (FIB) was placed in front of the coding sequence for green fluorescent protein or the active portion of the neuroactive peptide galanin (GAL). Subsequent studies showed that these vectors supported expression and constitutive secretion of these gene products from transfected cells in vitro. More importantly, upon transduction in vivo, AAV-FIB-GAL vectors significantly attenuated focal seizure sensitivity, and this seizure attenuation could be controlled in vivo by using a tetracycline-regulated promoter. The expression and constitutive secretion of green fluorescent protein, or the expression of GAL alone, exerted no effect on focal seizure sensitivity. Moreover, unilateral infusion of the AAV-FIB-GAL vectors into the hippocampus prevented kainic acid-induced hilar cell death. With regard to limbic seizures, bilateral infusion of AAV-FIB-GAL vectors into the piriform cortex prevented both behavioral and localized electrographic seizure activity after the peripheral administration of kainic acid. Also, when rats were electrically kindled to class V seizure activity, subsequent infusion of AAV-FIB-GAL proved capable of significantly elevating the seizure initiation threshold. Thus, these studies clearly demonstrate the anti-seizure effectiveness of AAV vector-mediated expression and constitutive secretion of galanin.

Secretion and Surface Display of Green Fluorescent Protein Using the Yeast Saccharomyces cerevisiae

Green fluorescent protein (GFP) continues to be a very useful tool in biotechnology, but soluble production of GFP and GFP-protein fusions has been difficult. In this study, we have produced yeast-enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a soluble, secreted product with a purified level of 6 mg/L. Expression was directed by the inducible GAL1-10 promoter and synthetic prepro leader sequence. The secretion of yEGFP by yeast was strongly dependent on temperature, with 20 degrees C induction being optimal. Use of 2 micro multicopy expression constructs elevated yields over a low-copy CEN-based system by approximately 2-fold. Yeast-enhanced GFP was also expressed as a fusion to the Aga2p mating agglutinin in order to test the secretory processing fidelity of yEGFP-protein fusions. When the cell surface anchoring protein, Aga1p, was co-overexpressed with the Aga2p-yEGFP fusion, the Aga2p-yEGFP protein was tethered to the yeast cell surface. Flow cytometry and fluorescence microscopy analysis indicated that the fusion was displayed on the yeast cell surface at high levels. In the absence of high level Aga1p expression, the Aga2p-yEGFP fusion protein was instead secreted in its entirety with no detectable surface display. These findings reveal that yeast is a suitable host for secretion of GFP and GFP-protein fusions and thus could enable a wide range of biochemistry and biotechnology applications.

Secretory delivery of heterologous proteins in attenuated Vibrio anguillarum for potential use in vaccine design

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP(empA) antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP(empA) antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.

형질전환된 벼세포배양에서 green fluorescent protein (GFP) 생산

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. In this study, GFP was produced and secreted from suspension cells derived from transgenic rice. The RAmy3E promoter placed before the GFP gene controlled by sugars such as sucrose. The effects of sucrose concentration on the secretion of GFP and total protein into the medium were investigated in batch suspension culture. It was possible, therefore, to induce the expression of the GFP by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. The dry cell weight (7.06 g/L) and GFP level were detected as highest at 12%, 3% sucrose after 20 day culture, respectively. However secreted GFP fluorescence at the other sucrose concentrations (6%, 12%, 18% and 24%) were a little amount in media.

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.

Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae α-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The α-factor signal did not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.

Regulated expression of green fluorescent protein in Debaryomyces hansenii.

The broad range of environmental conditions under which Debaryomyces hansenii can grow, and its production of lipolytic and proteolytic enzymes, have promoted its widespread use. The present work represents a preliminary characterization of D. hansenii for heterologous expression and secretion of green fluorescent protein (GFP). Six heterologous expression vectors were used to address protein production efficiency under regulated expression conditions. Protein expression in D. hansenii seems to be similar to that in Saccharomyces cerevisiae, with transcription being controlled by almost all of the S. cerevisiae and D. hansenii inducible promoters tested, with the exception of the alcohol dehydrogenase 2 gene promoter from S. cerevisiae. Extracellular protein levels in D. hansenii were lower than in S. cerevisiae when Saccharomyces signal peptides were used.

Secretion of foreign proteins mediated by chicken lysozyme gene regulatory sequences.

Exploitation of the insulating properties of the complete chicken lysozyme gene domain may facilitate the production of transgenic chicken bioreactors with the capacity to deposit valuable proteins in the egg white. Chimeric genes consisting of the chicken lysozyme gene regulatory sequences and sequences encoding foreign proteins could be inserted randomly into the chicken genome and retain appropriate expression levels. The research reported here established that chicken lysozyme gene regulatory sequences can be used to direct the production and secretion of green fluorescent protein (used as a reporter protein) in transiently transfected chicken blastodermal cells. Attempts to verify these findings in transgenic hens are currently in progress. To provide a rapid means of generating constructs encoding other foreign proteins under the control of lysozyme gene regulatory sequences that can facilitate the secretion of heterologous proteins in vivo, a generic lysozyme gene regulatory scaffold was created using a poxvirus-mediated gene targeting system.

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function.

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.

Mucin-like Domain of Enteropeptidase Directs Apical Targeting in Madin-Darby Canine Kidney Cells

Enteropeptidase, a type II transmembrane protein of the enterocyte brush border, is sorted directly to the apical membrane of Madin-Darby canine kidney II cells. Apical targeting appears to be mediated by an N-terminal segment that contains a 27-amino acid residue O-glycosylated mucin-like domain consisting of two short mucin-like repeats, A and B. Targeting signals within these repeats were characterized by using green fluorescent protein (GFP) as a reporter. Constructs with a cleavable signal peptide and both repeats A and B were secreted apically. Similar constructs lacking mucin repeats were secreted randomly. Either repeat A or B was sufficient to direct apical targeting of GFP. O-linked oligosaccharides alone were not sufficient for targeting because fusion to a different O-glycosylated motif did not alter the random secretion of GFP, and several constructs with mutations in either repeat A or B were O-glycosylated and secreted randomly. In addition, repeat B appears to contain an apical targeting signal that functions in the absence of glycosylation. Density gradient centrifugation indicated that, unlike several other apically targeted membrane and soluble proteins, apical sorting of mucin-GFP chimeric proteins does not appear to utilize lipid rafts.

Impediments to secretion of green fluorescent protein and its fusion from Saccharomyces cerevisiae.

While it has been demonstrated that GFP-tagged proteins were transported to their correct cellular compartments in most cells, attempts to secrete GFP/GFP-fusion through the default secretory pathway have not been as successful. In an attempt to induce secretion of GFP and Hexokinase (HXK)-GFP fusion in Saccharomycescerevisiae, we have cloned constructs that employed four different yeast secretion signal sequences, i.e., INU1, SUC2, PHO5, and MEL1. The expression is under the control of the galactose-inducible GAL1 promoter. Our results showed that all eight constructs entered the secretory pathway successfully, and the signal peptides were all cleaved off. However, none of the eight constructs were able to lead to secretion into the culture media or the periplasmic space. The expression levels of the eight constructs differ dramatically, depending on both the signal peptide and whether GFP was fused with HXK. Confocal microscopy studies revealed that the eight constructs also led to very different localization patterns. Among them, two constructs targeted GFP to the vacuole partially or exclusively, whereas others were mostly retained in the ER/Golgi compartments. Our efforts, together with those of others, seem to suggest that the signal peptide itself is not enough to lead to secretion of GFP from S. cerevisiae, although it has been successful in some other organisms. Nonetheless, the advantage of GFP's in vivo detection makes it a powerful tool for investigating protein localization events.

Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system.

Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.

Secretion of Green Fluorescent Protein from Recombinant Baculovirus-Infected Insect Cells

Trichoplusia ni(High Five) andSpodoptera frugiperda(Sf21) cells were engineered for expression of epitope (Flag)-tagged signal peptide-green fluorescent protein (GFP) fusions to examine the suitability of GFP as a secretory marker. The recombinant baculovirus-infected cells became fluorescent, and the High Five cells but not Sf21 cells secreted GFP in the culture medium as detected by the presence in the culture supernatant of a Flag-immunoreactive 30-kDa species and the characteristic 510-nm GFP fluorescence peak. Signal peptides derived from ecdysteroid UDP-glucosyltransferase ofAutographa californicanuclear polyhedrosis virus and from rat brain glutamate receptor were both able to promote secretion of GFP. GFP may thus be used as a research tool in the study of the secretory process in insect cells both in cell biology and in biotechnological applications.