Production Of Green Fluorescent Protein Research Articles

Cell‐free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care.

Development of SimCells as a novel chassis for functional biosensors

This work serves as a proof-of-concept for bacterially derived SimCells (Simple Cells), which contain the cell machinery from bacteria and designed DNA (or potentially a simplified genome) to instruct the cell to carry out novel, specific tasks. SimCells represent a reprogrammable chassis without a native chromosome, which can host designed DNA to perform defined functions. In this paper, the use of Escherichia coli MC1000 ∆minD minicells as a non-reproducing chassis for SimCells was explored, as demonstrated by their ability to act as sensitive biosensors for small molecules. Highly purified minicells derived from E. coli strains containing gene circuits for biosensing were able to transduce the input signals from several small molecules (glucarate, acrylate and arabinose) into the production of green fluorescent protein (GFP). A mathematical model was developed to fit the experimental data for induction of gene expression in SimCells. The intracellular ATP level was shown to be important for SimCell function. A purification and storage protocol was developed to prepare SimCells which could retain their functions for an extended period of time. This study demonstrates that SimCells are able to perform as ‘smart bioparticles’ controlled by designed gene circuits.

Potential microbial contamination during sampling of permafrost soil assessed by tracers

Drilling and handling of permanently frozen soil cores without microbial contamination is of concern because contamination e.g. from the active layer above may lead to incorrect interpretation of results in experiments investigating potential and actual microbial activity in these low microbial biomass environments. Here, we present an example of how microbial contamination from active layer soil affected analysis of the potentially active microbial community in permafrost soil. We also present the development and use of two tracers: (1) fluorescent plastic microspheres and (2) Pseudomonas putida genetically tagged with Green Fluorescent Protein production to mimic potential microbial contamination of two permafrost cores. A protocol with special emphasis on avoiding microbial contamination was developed and employed to examine how far microbial contamination can penetrate into permafrost cores. The quantity of tracer elements decreased with depth into the permafrost cores, but the tracers were detected as far as 17 mm from the surface of the cores. The results emphasize that caution should be taken to avoid microbial contamination of permafrost cores and that the application of tracers represents a useful tool to assess penetration of potential microbial contamination into permafrost cores.

Novel Detection Strategy To Rapidly Evaluate the Efficacy of Antichlamydial Agents.

Chlamydia trachomatis infections present a major heath burden worldwide. The conventional method used to detect C. trachomatis is laborious. In the present study, a novel strategy was utilized to evaluate the impact of antimicrobial agents on the growth of C. trachomatis and its expression of ompA promoter-driven green fluorescence protein (GFP). We demonstrate that this GFP reporter system gives a robust fluorescent display of C. trachomatis growth in human cervical epithelial cells and, further, that GFP production directly correlates to changes in ompA expression following sufficient exposure to antimicrobials. Validation with azithromycin, the first-line macrolide drug used for the treatment of C. trachomatis infection, highlights the advantages of this method over the traditional method because of its simplicity and versatility. The results indicate both that ompA is highly responsive to antimicrobials targeting the transcription and translation of C. trachomatis and that there is a correlation between changing GFP levels and C. trachomatis growth. This proof-of-concept study also reveals that the ompA-GFP system can be easily adapted to rapidly assess antimicrobial effectiveness in a high-throughput format.

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

Quorum Sensing Inhibition and Structure-Activity Relationships of β-Keto Esters.

Traditional therapeutics to treat bacterial infections have given rise to multi-drug resistant pathogens, which pose a major threat to human and animal health. In several pathogens, quorum sensing (QS)—a cell-cell communication system in bacteria—controls the expression of genes responsible for pathogenesis, thus representing a novel target in the fight against bacterial infections. Based on the structure of the autoinducers responsible for QS activity and other QS inhibitors, we hypothesize that β-keto esters with aryl functionality could possess anti-QS activity. A panel of nineteen β-keto ester analogs was tested for the inhibition of bioluminescence (a QS-controlled phenotype) in the marine pathogen Vibrio harveyi. Initial screening demonstrated the need of a phenyl ring at the C-3 position for antagonistic activity. Further additions to the phenyl ring with 4-substituted halo groups or a 3- or 4-substituted methoxy group resulted in the most active compounds with IC50 values ranging from 23 µM to 53 µM. The compounds additionally inhibit green fluorescent protein production by E. coli JB525. Evidence is presented that aryl β-keto esters may act as antagonists of bacterial quorum sensing by competing with N-acyl homoserine lactones for receptor binding. Expansion of the β-keto ester panel will enable us to obtain more insight into the structure–activity relationships needed to allow for the development of novel anti-virulence agents.

Enhancement of a high efficient autoinducible expression system in Bacillus subtilis by promoter engineering

Quorum-sensing related promoter srfA (PsrfA) was used to construct autoinducible expression system for production of recombinant proteins in Bacillus subtilis. PsrfA was prominent in the unique property of inducer-free activity that is closely correlated with cell density. Here, using green fluorescent protein (GFP) as the reporter protein, PsrfA was optimized by shortening its sequences and changing the nucleotides at the conserved regions of −35 −15 and −10 regions, obtaining a library of PsrfA derivatives varied in the strength of GFP production. Among all the promoter mutants, the strongest promoter P10 was selected and the strength in GFP expression was 150% higher than that of PsrfA. Heterologous protein of aminopeptidase and nattokinase could be overexpressed by P10, the activities of which were 360% and 50% higher than that of PsrfA, respectively. These results suggested that the enhanced promoter P10 could be used to develop autoinducible expression system for overexpression of heterologous proteins in B. subtilis.

Framework for Kriging‐based iterative experimental analysis and design: Optimization of secretory protein production in Corynebacterium glutamicum

The production of bulk enzymes used in food industry or organic chemistry constitutes an important part of industrial biotechnology. The development of production processes for novel proteins comprises a variety of biological engineering and bioprocess reaction engineering factors. The combinatorial explosion of these factors can be effectively countered by combining high‐throughput experimentation with advanced algorithms for data analysis and experimental design. We present an experimental optimization strategy that merges three different techniques: (1) advanced microbioreactor systems, (2) lab automation, and (3) Kriging‐based experimental analysis and design. This strategy is demonstrated by maximizing product titer of secreted green fluorescent protein (GFP), synthesized by Corynebacterium glutamicum, through systematic variation of CgXII minimal medium composition. First, relevant design parameters are identified in an initial fractional factorial screening experiment. Then, the functional relationship between selected media components and protein titer is investigated more detailed in an iterative procedure. In each iteration, Kriging interpolations are used for formulating hypotheses and planning the next round of experiments. For the optimized medium composition, GFP product titer was more than doubled. Hence, Kriging‐based experimental analysis and design has been proven to be a powerful tool for efficient process optimization.

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.

BackgroundBacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA).ResultsThe promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the −10 and −35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level.ConclusionThe auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins.

Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein

Cellulase is a widely used enzyme for saccharification of plant cell walls for the production of biofuels. In our work, we tested an ethanol inducible native alcA promoter from Aspergillus nidulans in Escherichia coli (E. coli) and tobacco. The alcA promoter was fused with a cellulase gene and then the promoter was compared with other widely used promoters, namely, T5 and cauliflower mosaic virus (CaMV) 35S in E. coli. We also tested the activity of alcA promoter in tobacco by transiently expressing a green fluorescent protein gene under the control of alcA promoter. We concluded from our quantitative polymerase chain reaction (qPCR) results that when alcA promoter was expressed along with AlcR transcription factor, the alcA promoter was 11 times more active than the T5 promoter. The qPCR data suggested that there may be a direct correlation between AlcR concentrations and alcA activity, indicating a key role of the activator AlcR in the regulation of the alcA promoter. Our work on inducible promoter system for cellulase production may open new avenues of research in the field of biofuel development from living cells.

Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium.

The Gram-positive bacterium Bacillus megaterium was systematically developed for the plasmid-based production of recombinant proteins at the gram-per-liter scale. The amount of protein produced per cell was found strongly correlated to the codon usage of the heterologous gene of interest in comparison to the codon usage of B. megaterium. For analyzing the influence of rare codons on the translational efficiency and protein production in B. megaterium, a test system using the gene for the green fluorescent protein (GFP) as reporter was established. For this purpose, four consecutive identical codons were introduced into the 5' end of gfp and the resulting variations in GFP formation were quantified. Introduction of the rare codons GCC, CGG, and ACC for alanine, arginine, and threonine reduced GFP production 2.1-, 3.3-, and 1.7-fold in comparison to the favored codons GCU, CGU, and ACA, respectively. Coexpression of the corresponding rare codon tRNA (rctRNA) genes improved GFP production 4.2-, 2.7-, and 1.7-fold, respectively. The system was applied to the production of a formate dehydrogenase (FDH) from Mycobacterium vaccae and an extracellular hydrolase (TFH) from Thermobifida fusca. Coexpression of one to three different rctRNA genes resulted in an up to 18-fold increased protein production. Interestingly, rctRNA gene coexpression also elevated the production of M. vaccae FDH and T. fusca TFH from codon optimized genes, indicating a general positive effect by rctRNA gene overexpression on the protein production in B. megaterium. Thus, the basis for a B. megaterium enhanced production strain coexpressing rctRNA genes was laid.

Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies.

Synthetic gene involving azobenzene-tethered T7 promoter for the photocontrol of gene expression by visible light.

In the present study, we demonstrate photoregulation of gene expression in a cell-free translation system from a T7 promoter containing two azobenzene derivatives at specific positions. As photoswitches, we prepared azobenzene-4'-carboxlyic acid (Azo) and 2,6-dimethylazobenzene-4'-carboxylic acid (DM-Azo), which were isomerized from trans to cis upon irradiation with UV light (λ < 370 nm), and 4-methylthioazobenzene-4'-carboxylic acid (S-Azo) and 2,6-dimethyl-4-(methylthio)azobenzene-4'-carobxylic acid (S-DM-Azo), which were cis-isomerized by irradiation with 400 nm visible light. Expression of green fluorescent protein from a promoter modified with S-Azo or S-DM-Azo could be induced by harmless visible light whereas that from a promoter modified with Azo or DM-Azo was induced only by UV light (340-360 nm). Thus, efficient photoregulation of green fluorescent protein production was achieved in a cell-free translation system with visible light without photodamage.

Production of recombinant protein in Escherichia coli cultured in extract from waste product alga, Ulva lactuca.

This study examined the potential for waste product alga, Ulva lactuca, to serve as a media component for recombinant protein production in Escherichia coli. To facilitate this investigation, U. lactuca harvested from Jamaica Bay was dried, and nutrients acid extracted for use as a growth media. The E. coli cell line BL21(DE3) was used to assess the effects on growth and production of recombinant green fluorescent protein (GFP). This study showed that media composed of acid extracts without further nutrient addition maintained E. coli growth and recombinant protein production. Extracts made from dried algae lots less than six-months-old were able to produce two-fold more GFP protein than traditional Lysogeny Broth media.

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.

Effect of Bavachinin and its derivatives on T cell differentiation

Bavachinin, which can be isolated from the Chinese herb Fructus Psoraleae, has the potential as a potent anti-asthma drug. However, the extremely low water solubility of Bavachinin limits its application. In this study, two new derivatives of Bavachinin, i.e., compounds A and B, whose water solubility is better than that of Bavachinin, were synthesized via biotransformation. A comparative investigation was then performed on the effects of these two new derivatives, along with Bavachinin, on T cell differentiation. The results showed that they have different effects. Bavachinin and compound B inhibited green fluorescent protein (GFP) production from the T cells of IL-4-GFP-enhanced transcript (4GET) mice, whereas compound A did not. The effect was mainly attributed to the inhibition of GATA-3 protein production. Bavachinin and compound B can inhibit the production of GATA-3 mRNA, but they showed different effects on the production of T-bet mRNA. Compound B increased the production of T-bet mRNA, whereas Bavachinin did not. The results will be very useful for optimizing Bavachinin so that potent anti-allergic drugs can be developed. The structure-activity relationship of Th2 was revealed based on the difference between Bavachinin and compound B. This finding can enrich the database of preliminary drug screening from their chemical structures.

Role of endocytic uptake in transfection efficiency of solid lipid nanoparticles‐based nonviral vectors

As has been shown for different vector systems, the entry pathway(s) impacts upon the transfection efficiency. The present study aimed to explore the cellular uptake mechanisms of three different vectors based on solid lipid nanoparticles (SLN) in HeLa cells. The use of endocytosis inhibitors that affect specific internalization pathways provides a tool for the study of these routes. We prepared three vectors based on solid lipid nanoparticles: without protamine, with protamine, and with protamine and dextran. Uptake, percentage of transfected HeLa cells and enhanced green fluorescent protein (EGFP) production were all analyzed in the presence or absence of different endocytosis inhibitors. In addition, co-localization studies using lysosomal markers were carried out to determine the influence of the trafficking to late endosomal compartments on the transfection capacity of the vectors. Uptake and transfection of each vector was affected differently by each endocytosis inhibitor. Ethylisopropylamiloride (EIPA) did not affect uptake of the DNA-SLN vector, whereas all of the inhibitors affected transfection. In the case of protamine-DNA-SLN and dextran-protamine-DNA-SLN vectors, EIPA affected uptake and dynasore did not decrease transfection. DNA-SLN vector appear to enter productively by multiple pathways in HeLa cells. By contrast, dynamin does not appear to be essential in the productive entry of protamine-containing vectors. In addition, enhancement of the macropinocytic route increases EGFP production when dextran is added to the vector.

Metabolic flux distribution and thermodynamic analysis of green fluorescent protein production in recombinant Escherichia coli: The effect of carbon source and CO 2 partial pressure

Increasing recombinant protein production yields from bacterial cultures remains an important challenge in biotechnology. Acetate accumulation due to high dissolved carbon dioxide (pCO2) concentrations in the medium has been identified as a factor that negatively affects such yields. Under appropriate culture conditions, acetate could be re-assimilated by bacterial cells to maintain heterologous proteins production. In this work, we developed a simplified metabolic network aiming to establish a reaction rate analysis for a recombinant Escherichia coli when producing green fluorescent protein (GFP) under controlled pCO2 concentrations. Because E. coli is able to consume both glucose and acetate, the analysis was performed in two stages. Our results indicated that GFP synthesis is an independent process of cellular growth in some culture phases. Additionally, recombinant protein production is influenced by the available carbon source and the amount of pCO2 in the culture medium. When growing on glucose, the increase in the pCO2 concentration produced a down-regulation of central carbon metabolism by directing the carbon flux toward acetate accumulation; as a result, cellular growth and the overall GFP yield decreased. However, the maximum specific rate of GFP synthesis occurred with acetate as the main available carbon source, despite the low activity in the other metabolic pathways. To maintain cellular functions, including GFP synthesis, carbon flux was re-distributed toward the tricarboxylic acid cycle and the pentose phosphate pathway to produce ATP and NADH. The thermodynamic analysis allowed demonstrating the feasibility of the simplified network for describing the metabolic state of a recombinant system.

Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots

A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.

Genetic engineering ofLactobacillus diolivorans

In this study, we developed a toolbox for genetic manipulation of Lactobacillus diolivorans, a promising production organism for 1,3-propanediol from glycerol. Two major findings play a key role for successful transformation of this organism: (1) the absence of a native plasmid, because a native plasmid is a major obstacle for transformation of L.diolivorans, and (2) the absence of DNA methylation. A suitable expression plasmid, pSHM, for homologous and heterologous protein expression in L.diolivorans was constructed. This plasmid is based on the replication origin repA of L.diolivorans. The native glyceraldehyde-3-phosphate dehydrogenase promoter is used for constitutive expression of the genes of interest. Functional expression of genes in L.diolivorans was shown with two examples: production of green fluorescent protein resulted in a 40- to 60-fold higher fluorescence of the obtained clones compared with the wild-type strain. Finally, the homologous overexpression of a putatively NADPH-dependent 1,3-propanediol oxidoreductase improved 1,3-propanediol production by 20% in batch cultures.