Expression Level Of Recombinant Protein Research Articles

Enhanced expression and purification of anti-VEGF nanobody in cucurbit plants

Neutralizing nanobodies against vascular endothelial growth factor (VEGF) and its receptors are the most important types of medicine which are used in cancer therapy strategies. To increase the level of anti-VEGF (Nb42) nanobody accumulation and also to simplify its subsequent purification, we fused codon-optimized Nb42 nanobody to hydrophobin I (HFBI) stabilizing partner, and transiently expressed the fusion gene cassette in cucurbit plants using a zucchini yellow mosaic virus-based viral vector. The successful transcription of the target gene was confirmed with the use of RT-PCR and Real-time PCR analyses. According to the immunoblot assay results the expression level of Nb42-HFBI fusion protein, 22 μg/g fresh leaf tissue, was twofolds higher than the unfused nanobody. Furthermore, the endoplasmic reticulum (ER)-targeted Nb42-HFBI fusion protein was successfully purified from leaf extract using an aqueous-two phase system (ATPS) method. The use of HFBI fusion partner increased the expression level of the Nb42 recombinant protein while simultaneously facilitated its subsequent purification using an inexpensive non-chromatographic method known as ATPS. As a conclusion, the application of ER-targeted hydrophobin fusion strategy with the use of special features of plant viral vectors, can improve the value of plant-based transient expression platform for the production of important proteins with the industrial and therapeutic application.

Efficient increase of the novel recombinant human plasminogen activator expression level and stability through the use of homozygote transgenic rabbits.

Expression efficacy of recombinant protein in current expression systems is generally low. Therefore, the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this, the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammary‑specific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzyme‑linked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits, while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition, the expression of rhPA in homozygous transgenic rabbit was ~950µg/ml, which was markedly higher in comparison with that in hemizygote rabbits. Furthermore, increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.

Abstract 5653: VMax™: A new platform for recombinant protein expression

Abstract Here we describe Vmax™, a next-generation protein expression platform based on the organism V. natriegens, an extremely rapidly growing gram-negative prokaryote capable of reaching high cell density. Vmax™ contains an IPTG inducible T7 expression system, enabling high levels of recombinant protein expression. By combining a robust expression system with a highly productive organism, Vmax allows researchers to perform experiments faster, shorten workflow times, and generate large amounts of recombinant protein. Citation Format: Eric Hesek, Chris Wilson, Lindsey Wolf, Sanjay Vasu, Michael Sturges, Daniel Gibson, Matthew Weinstock. VMax™: A new platform for recombinant protein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5653.

Microbial production of toxins from the scorpion venom: properties and applications.

Scorpion venom are composed mainly of bioactive proteins and peptides that may serve as lead compounds for the design of biotechnological tools and therapeutic drugs. However, exploring the therapeutic potential of scorpion venom components is mainly impaired by the low yield of purified toxins from milked venom. Therefore, production of toxin-derived peptides and proteins by heterologous expression is the strategy of choice for research groups and pharmaceutical industry to overcome this limitation. Recombinant expression in microorganisms is often the first choice, since bacteria and yeast systems combine high level of recombinant protein expression, fast cell growth and multiplication and simple media requirement. Herein, we present a comprehensive revision, which describes the scorpion venom components that were produced in their recombinant forms using microbial systems. In addition, we highlight the pros and cons of performing the heterologous expression of these compounds, regarding the particularities of each microorganism and how these processes can affect the application of these venom components. The most used microbial system in the heterologous expression of scorpion venom components is Escherichia coli (85%), and among all the recombinant venom components produced, 69% were neurotoxins. This review may light up future researchers in the choice of the best expression system to produce scorpion venom components of interest.

Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences.

The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single‐molecule‐based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway® system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5′‐UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5′‐UTR.

Transient Recombinant Protein Production in Glycoengineered Nicotiana benthamiana Cell Suspension Culture.

Transient recombinant protein production is a promising alternative to stable transgenic systems, particularly for emergency situations in which rapid production of novel therapeutics is needed. In plants, Agrobacterium tumefaciens can be used as a gene delivery vector for transient expression. A potential barrier for plant-based production of human therapeutics is that different glycosylation patterns are found on plant and mammalian proteins. Since glycosylation can affect the efficacy, safety and stability of a therapeutic protein, methods to control glycan structures and distributions in plant-based systems would be beneficial. In these studies, we performed Agrobacterium-mediated transient expression in glycoengineered plant cell suspension cultures. To reduce the presence of plant-specific glycans on the product, we generated and characterized cell suspension cultures from β-1,2-xylosyltransferase and α-1,3-fucosyltransferase knockdown Nicotiana benthamiana. An anthrax decoy fusion protein was transiently produced in these glycoengineered plant cell suspension cultures through co-culture with genetically engineered Agrobacterium. The mass ratio of Agrobacterium to plant cells used was shown to impact recombinant protein expression levels. N-glycosylation analysis on the anthrax decoy fusion protein produced in glycoengineered N. benthamiana showed a dramatic reduction in plant-specific N-glycans. Overall, the results presented here demonstrate the feasibility of a simple, rapid and scalable process for transient production of recombinant proteins without plant-specific glycans in a glycoengineered plant cell culture host.

Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants

Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on protein production and provide evidence that N-glycosylation greatly increases the expression levels of ER-targeted recombinant proteins. Fusion of the extracellular domain (M domain) of protein tyrosine phosphatase receptor type C (CD45), which contains four putative N-glycosylation sites to a model protein, leptin at the C-terminus, increased recombinant protein levels by 6.1 fold. This increase was specific to ER-targeted proteins and was dependent on N-glycosylation. Moreover, expression levels of leptin, leukemia inhibitory factor and GFP were also greatly increased by fusion of M domain at either the N or C-terminus. Furthermore, the increase in protein levels resulted from enhanced translation, but not transcription. Based on these results, we propose that fusing a small domain containing N-glycosylation sites to target proteins is a powerful technique for increasing the expression levels of recombinant proteins in plants.

DESIGN OF HEPATITIS E VIRUS GENOTYPE 1 RECOMBINANT ORF3 PROTEIN BY CODON OPTIMIZATION METHOD

Aim. The development of the hepatitis E virus (HEV) genotype 1 full-size ORF3 recombinant polypeptide. Materials and methods. Escherichia coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods. Results. HEV genotype 1 RNA had been isolated from clinical samples collected in Kyrgyzstan. DNA copy of subgenomic virus RNA had been cloned and used for further development of E.coli strains producing full-size recombinant protein ORF3 fused to E.coli beta-galactosidase. Codons optimization method was used in aim to increase expression level of recombinant protein. Recombinant protein ORF3 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. Antigenic specificity of recombinant polypeptide had been confirmed by enzyme-linked immunosorbent assay and Western blotting with the specific sera. Conclusion. HEVgenotype 1 ORF3 recombinant antigen had been designed, and it’s applicability in diagnostic tests had been experimentally confirmed.

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica

BackgroundAn inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous.ResultsIn this study, we evaluate a number of promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5′ fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system.ConclusionsOur studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.

Novel mRNA-specific effects of ribosome drop-off on translation rate and polysome profile.

The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5’ to the 3’ end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling studies.

BioSpotlight / Citations

BioTechniquesVol. 62, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114530AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Stabler expression in stable cell linesA major issue with stable cell lines generated by standard methods is the heterogeneous levels of recombinant protein expression in different cell clones due to the integration of variable numbers of the transgene and/or position effects caused by integration of the transgene into random locations in the genome. In addition, the use of strong heterologous promoters in transgenic vectors can drive expression to very high, non-physiological levels that harm the cells. In this month's issue, researchers led by Brian Chen at McGill University describe an efficient method for stable cell line generation that achieves controlled, physiological expression of a transgene. Using CRISPR/Cas9 gene editing, the team inserted a single copy of a transgene at a specific gene locus, placing it under the control of an endogenous promoter with the desired level of gene expression. Their transgenic vector also includes a protein quantitation reporter gene to produce an equimolar amount of a fluorescent protein, allowing easy quantitation of the recombinant protein.See “Generating stable cell lines with quantifiable protein production using CRISPR/Cas9 mediated knock-in” on page 165.Two colors are better than one for dna methylation assayThe cytosine extension assay (CEA) assesses global DNA methylation levels by tracking the methylation of a CpG-containing restriction enzyme site, for example CCGG, which when methylated cannot be digested by HpaII but can be digested by its isoschizomer, MspI. The relative digestion of CCGG sites in a DNA sample by HpaII compared to MspI reflects the extent of DNA methylation and is assessed in CEA by the incorporation of a fluorescently labeled cytosine through end-filling by DNA polymerase of the 5′ guanosine overhang at the HpaII/MspI cut site. In this issue of BioTechniques, Craig Parfett and his colleagues at Health Canada present an improved version of CEA. In standard CEA, the same fluorescently colored cytosine is used in the extension reactions following HpaII and MspI digestion. In their version, Parfett's team used a different fluorescently colored cytosine for the extension reaction of each digest, after which the reactions were combined for downstream purification and signal detection. This two-color CEA showed greater precision compared to the one-color CEA and is well-suited to high-throughput screening.See “Two-color fluorescent cytosine extension assay for the determination of global DNA methylation” on page 157.Root problemsPlant biologists have long been interested in understanding the interactions that occur in nature between microorganisms and plant roots. Plants both obtain information about their environment and send out signals to microbes through their roots. However, deciphering the interplay between plant roots and the microbial communities surrounding them has proven difficult. Now, researchers report in PNAS a new microfluidic-based device designed specifically for monitoring root–bacteria interactions over time. The device, which the authors named the tracking root interaction system (TRIS), is composed of 9 independent microfluidic chambers where roots can be established and into which bacteria are introduced for monitoring. The chambers are coupled to a microscope assembly that makes it possible to obtain both spatial and temporal information on root–bacteria interactions. In proof-of-concept experiments, the authors fluorescently labeled bacteria and were able to observe root colonization in real time over extended periods using their TRIS device.Massalha, H. et al. 2017. Live imaging of root-bacteria interactions in a microfluidics setup. Proc Natl Acad Sci USA. doi:10.1073/pnas.1618584114.Western blotting goes smallWestern blotting is a commonly used technique for studying cellular proteins. While effective when using large numbers of cells as starting material, western blot analysis has proven more difficult when scaled down for studies of single cells with their much lower protein concentrations. Seeking to overcome this limitation and extend the range of protein analysis, researchers describe a new rare-cell, single-cell western blotting procedure in Nature Communications. Building on previous work that established the feasibility of single-cell resolution western blotting, the authors sought to measure panels of proteins from extremely rare circulating tumor cell (CTC) isolates. For protein analysis, each CTC was placed into an individual microwell, where the cell was first lysed and then immediately electrophoresed into a polyacrylamide matrix. Following a miniaturized version of polyacrylamide gel electrophoresis (PAGE), in-gel immunoprobing was done on the size-separated CTC proteins. Using this method, the authors were able to successfully perform multiplexed protein analyses and report protein expression on a per-cell basis.Sinkala, E. et al. 2017. Profiling protein expression in circulating tumor cells using microfluidic western blotting. Nat Commun. 8:14622.FiguresReferencesRelatedDetails Vol. 62, No. 4 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print April 2017 Information© 2017 Author(s)PDF download

Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris

The alcohol oxidase 1 promoter (PAOX1) of Pichia pastoris is commonly used for high level expression of recombinant proteins. While the safety risk of methanol and tough process control for methanol induction usually cause problems especially in large-scale fermentation. By testing the functions of trans-acting elements of PAOX1 and combinatorially engineering of them, we successfully constructed a methanol-free PAOX1 start-up strain, in which, three transcription repressors were identified and deleted and, one transcription activator were overexpressed. The strain expressed 77% GFP levels in glycerol compared to the wide-type in methanol. Then, insulin precursor (IP) was expressed, taking which as a model, we developed a novel glucose-glycerol-shift induced PAOX1 start-up for this methanol-free strain. A batch phase with glucose of 40 g/L followed by controlling residual glucose not lower than 20 g/L was compatible for supporting cell growth and suppressing PAOX1. Then, glycerol induction was started after glucose used up. Accordingly, an optimal bioprocess was further determined, generating a high IP production of 2.46 g/L in a 5-L bioreactor with dramatical decrease of oxygen consumption and heat evolution comparing with the wild-type in methanol. This mutant and bioprocess represent a safe and efficient alternative to the traditional glycerol-repressed/methanol-induced PAOX1 system.

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Degeneracy in the genetic code allows multiple codon sequences to encode the same protein. Codon usage bias in genes is the term given to the preferred use of particular synonymous codons. Synonymous codon substitutions had been regarded as "silent" as the primary structure of the protein was not affected; however, it is now accepted that synonymous substitutions can have a significant effect on heterologous protein expression. Codon optimization, the process of altering codons within the gene sequence to improve recombinant protein expression, has become widely practised. Multiple inter-linked factors affecting protein expression need to be taken into consideration when optimizing a gene sequence. Over the years, various computer programmes have been developed to aid in the gene sequence optimization process. However, as the rulebook for altering codon usage to affect protein expression is still not completely understood, it is difficult to predict which strategy, if any, will design the "optimal" gene sequence. In this review, codon usage bias and factors affecting codon selection will be discussed and the evidence for codon optimization impact will be reviewed for recombinant protein expression using plants as a case study. These developments will be relevant to all recombinant expression systems; however, molecular pharming in plants is an area which has consistently encountered difficulties with low levels of recombinant protein expression, and should benefit from an evidence based rational approach to synthetic gene design. Biotechnol. Bioeng. 2017;114: 492-502. © 2016 Wiley Periodicals, Inc.

Tunable recombinant protein expression in E. coli: enabler for continuous processing?

Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in Escherichia coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death, and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell’s metabolic capacities to a maximum. Within this article, we review genetic and process technological aspects of tunable expression systems in E. coli, providing a roadmap for the industrial exploitation of the reviewed technologies. We attempt to differentiate the term “expression tuning” from its inflationary use by providing a concise definition and highlight interesting fields of application for this versatile new technology. Dependent on the type of inducer (metabolizable or non-metabolizable), different process strategies are required in order to achieve tuning. To fully profit from the benefits of tunable systems, an independent control of growth rate and expression rate is indispensable. Being able to tackle problems such as long-term culture stability and constant product quality expression tuning is a promising enabler for continuous processing in biopharmaceutical production.

Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology.

BackgroundRecombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor affecting mRNA concentration can alter the protein expression level. Response surface methodology (RSM) based on the Box-Behnken design (BBD) is a statistical tool for experimental design and for optimizing biotechnological processes.ObjectivesThis investigation aimed to predict and develop the optimal culture conditions for mRNA expression of the synthetic human endostatin (hES) gene in Escherichia coli BL21 (DE3).Materials and MethodsThe hES gene was amplified, cloned, and expressed in the E. coli expression system. Three factors, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, post-induction time, and cell density before induction, were selected as important factors. The mRNA expression level was determined using real-time PCR. The expression levels of hES mRNA under the different growth conditions were analyzed. SDS-PAGE and western blot analyses were carried out for further confirmation of interest-gene expression.ResultsA maximum rhES mRNA level of 376.16% was obtained under the following conditions: 0.6 mM IPTG, 7 hours post-induction time, and 0.9 cell density before induction. The level of rhES mRNA was significantly correlated with post-induction time, IPTG concentration, and cell density before induction (P < 0.05). The expression of the hES gene was confirmed by western blot.ConclusionsThe obtained results indicate that RSM is an effective method for the optimization of culture conditions for hES gene expression in E. coli.

High-level expression of prolyl endopeptidase in Pichia pastoris using PLA2 as a fusion partner

In our previous studies, a prolyl endopeptidase (PEP) gene from Aspergillus oryzae (MOH) was cloned and expressed in Pichia pastoris; however, the recombinant protein expression level of MOH was very low. In the present study, the PEP expression level was successfully improved by constructing fusion expression proteins with four fusion partners, namely, Streptomyces violaceoruber Phospholipase A2 (PLA2), cellulose-binding domain (CBD), small ubiquitin-related modifier (SUMO) and maltose binding protein (MBP). The enzyme activities of the recombinant fusion proteins CLMH, SLMH, MLMH and PLMH were increased to 3.8-, 2.7-, 4.9- and 7.4-fold compared with that of the parent MOH. Moreover, the extracellular protein content of CLMH, SLMH, MLMH, PLMH were 1.42-, 1.25-, 1.67- and 1.83-fold higher compared with that of MOH. Both PLMH and MOH showed the highest activity at pH 5.5, the highest stability at pH 6.0 and maximal activity at 40°C.

Genetic analysis of the clonal stability of Chinese hamster ovary cells for recombinant protein production.

Chinese hamster ovary (CHO) cells are frequently used for the production of recombinant proteins for therapeutical applications. However, the recombinant protein expression level of CHO cells may reduce during long-term culture. The physiological changes related to the stability of expression were not well understood. In this study, we performed a series of genetic analysis on stable and unstable clonal derived populations. Transcriptome analysis revealed that a large number of differentially expressed genes (>100) were identified in the unstable population between early and late generations, while only a few differentially expressed genes were found in the stable population, suggesting that the gene expression change is related to the instability of recombinant protein production. On the other hand, no significant differences were found in promoter methylation or gene copy numbers in the unstable population. Taken together, our data help better understand the molecular mechanism underlying the stability of recombinant protein production in CHO cells.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

A Comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq.

BackgroundSoybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene.MethodsEach transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting. Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing. Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks. Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO.ResultsThe transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected.ConclusionsMeasurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0207-z) contains supplementary material, which is available to authorized users.

Regulatory Elements in Vectors for Efficient Generation of Cell Lines Producing Target Proteins

To date, there has been an increasing number of drugs produced in mammalian cell cultures. In order to enhance the expression level and stability of target recombinant proteins in cell cultures, various regulatory elements with poorly studied mechanisms of action are used. In this review, we summarize and discuss the potential mechanisms of action of such regulatory elements.