Finding one of a kind: advances in single-protein production

An ultimate goal for any protein production system is to express only the protein of interest without producing other cellular proteins. To date, there are only two established methods that will allow the successful expression of only the protein of interest: the cell-free in vitro protein synthesis system and the in vivo single-protein production (SPP) system. Although single-protein production can be achieved in cell-free systems, it is not easy to completely suppress the production of cellular proteins during the production of a protein of interest in a living cell. However, the finding of a unique sequence-specific mRNA interferase in Escherichia coli led to the development of the SPP system by converting living cells into a bioreactor that produces only a single protein of interest without producing any cellular proteins. This technology not only provides a new high expression system for proteins, but also offers a novel avenue for protein structural studies.     

Single protein production (SPP) system in Escherichia coli

Here, we provide a detailed protocol for the single protein production (SPP) system, which is designed to produce only a single protein of interest in living Escherichia coli cells. Induction of MazF, an mRNA interferase that cleaves RNA at ACA nucleotide sequences, results in complete cell growth arrest. However, if mRNA encoding a protein of interest is engineered to be devoid of ACA base triplets and is induced at 15 degrees C using pCold vectors in MazF-expressing cells, only the protein from this mRNA is produced at a yield of 20-30% of total cellular protein; other cellular protein synthesis is almost completely absent. In theory, any protein can be produced by the SPP system. Protein yields are typically unaffected even if the culture is condensed up to 40-fold, reducing the cost of protein production by up to 97.5%. The SPP system has a number of key features important for protein production, including high-yield and prolonged production of isotope-labeled protein at a very high signal-to-noise ratio. The procedure can be completed in 7 d after cloning of an ACA-less target gene into the expression system.     

Multi-Host Expression System for Recombinant Production of Challenging Proteins

Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class.     

单蛋白合成系统研究进展

任何一个蛋白质合成系统的最终目标都是表达所需要的蛋白质(目的蛋白质)而不产生其他的细胞蛋白质。目前,只有两种方法可以成功的表达目的蛋白质:体外无细胞蛋白质合成系统和体内单蛋白生产(SPP)系统。综述现今不同的单蛋白生产系统,讨论他们的应用、优点和缺点。     

Net protein contribution and enteric methane production of pasture and grain-finished beef cattle supply chains

Ruminant red meat production systems around the world often include a grain feeding phase. The role of red meat in the food system is therefore often discussed in terms of the food vs feed debate, as well as invoking the comparatively poor feed conversion efficiency of ruminants and climate impacts from enteric methane. The concept of net protein contribution (NPC) incorporates the quality attributes of protein produced by livestock systems into estimates of the efficiency of production systems. We applied the NPC method to two Australian beef supply chains, i) Grass-fed and ii) Grain-finished beef, using an established model of ruminant grazing systems (GrassGro®) and these are reflective of beef production systems in other countries. The beef supply chains evaluated did not compete with humans for protein. The Grain-finished beef supply chain, while positively contributing to human protein requirements (NPC value 1.96), had markedly lower NPC values than the Grass-fed system (NPC value 1 597). However, Grass-fed beef production systems have a higher methane intensity than the Grain-finished supply chain. The two examples of pasture-based beef production systems examined provide a positive net protein contribution to human food supply, even with extended periods of finishing on grain-based diets. This is achieved by ruminant grazing on pastures converting low-quality forage into high value human edible protein. The efficiency of protein production varies according to the system design, and other considerations such as land use and enteric methane production are elements that need consideration in the overall assessment of the production footprint.     

Automated system for high-throughput protein production using the dialysis cell-free method

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14 h, including 8 h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).     

Independently inducible system of gene expression for condensed single protein production (cSPP) suitable for high efficiency isotope enrichment

The ability to produce isotope-enriched proteins is fundamental to the success of modern protein NMR, and is particularly essential for NMR activities in structural genomics projects. Conventional methods of protein production often prove to be cost prohibitive for obtaining samples, particularly perdeuterated and site-specifically labeled proteins. The condensed single protein production system (cSPP), providing protein expression following condensation of cells 10–40 fold, allows for the production of such samples at a fraction of the cost. The previously described cSPP system is a two plasmid system where both the MazF toxin and ACA-less target gene are coinduced with IPTG. Coinduction results in 10–20% of the target protein produced without isotopic enrichment. Though the unlabeled protein is generally not visible in isotope-filtered NMR experiments, it results in an effective reduction in yield of the observable sample. By altering the cSPP system and separating the induction of the MazF toxin, required to convert cells into a semiquiescent state prior to condensation, from the expression of the target gene, we are now able to eliminate the unlabeled protein fraction and improve the isotope incorporation. Here we describe a series of pCold(tet) vectors with various features that can be used in the dual inducible cSPP(tet) system to obtain high-quality isotopically enriched protein at as little as 2.5% the cost of traditional methods.     

Genetic manipulation of stationary-phase genes to enhance recombinant protein production in Escherichia coli

Genetic manipulation of the host strain, by which cell physiology could be modulated, was exploited to enhance recombinant protein production in Escherichia coli. The effects of an inactivated stationary-phase gene (rmf or katF) on recombinant protein production in strains with two different expression systems (the pH-inducible and the lac promoters) were investigated. An improvement of recombinant protein production in the katF mutant at low growth rates was observed for both expression systems. A fourfold and a 30% increase in the volumetric recombinant protein activity were observed for the pH-inducible and the lac promoter system, respectively. The effect of the rmf mutation, on the other hand, depends on the expression system. A twofold increase in the volumetric recombinant protein activity was found for the pH-inducible promoter system, but there was no improvement for the lac promoter system. Improvement in culture performance for slow-growing cultures may have an impact on the design strategy of the host/vector system used in fed-batch cultures, where the specific growth rate is usually slow. The information may also be useful for developing optimal host/vector gene expression systems for recombinant protein production. (c) 1996 John Wiley & Sons, Inc.     

Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system

The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.     

Effective approaches for the production of heterologous proteins using the Thermococcus kodakaraensis-based translation system

We have previously established a system for cell-free protein synthesis that can be operated at high temperatures using the cell lysate of a hyperthermophilic archaeon, Thermococcus kodakaraensis. To apply this system to practical use in the field of heterologous protein production, the performance of our system in the synthesis of green fluorescent protein (GFP) was examined. As the wild-type GFP is a thermolabile protein, a thermostable GFP derivative (tGFP) was selected as a candidate for protein synthesis. The first attempt of tGFP synthesis at 60 degrees C using the system resulted in a detection of small amount of protein (<0.1 microg/mL) by Western blot analysis. Using a newly synthesized tGFP gene in which codon usage was optimized for T. kodakaraensis as a template, tGFP was clearly detectable at temperatures between 50 and 65 degrees C. The tGFP production was further enhanced over 10 microg/mL with the addition of stem-loop structure at the 3'-end of mRNA. Determination of fluorescences of tGFP in the reaction mixtures indicated that active tGFP constituted ca. 30% of the total protein synthesized. Addition of T. kodakaraensis chaperonin to the system significantly increased the ratio of active tGFP content to ca. 50%. Through these approaches to the system, the production of tGFP increased over 100-fold, and the yield of active tGFP synthesized reached to 6.5 microg/mL.     

Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.     

Characterization of the stability of recombinant protein production in the GS-NS0 expression system

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.     

转基因植物表达重组蛋白的研究进展

植物表达系统的一些潜在优点 ,如重组蛋白的高积累水平 ,糖基化 ,细胞内的定位和自然储藏的稳定性是目前植物生产重组蛋白系统研究成为热点的主要原因 .在研究和选择转基因植物表达系统的过程中 ,转化 ,转化后 ,翻译 ,翻译后等环节都会影响到最终产物的数量和质量 ,因此应该了解基因表达的规律 ,以制定植物生产重组蛋白合适的策略 ,重组蛋白积累水平是关键 ,但其它因素如植物的选择 ,转基因植物的处理 ,下游加工等同样重要 .某些情形下 ,仅下游加工的成本一项就影响到特定植物表达系统的实际应用价值 .     

Two‐compartment processing as a tool to boost recombinant protein production

Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol‐induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one‐compartment system cannot boost productivity for the model system under study. Hence, a two‐compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen‐related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two‐fold increase in the titer and productivity and three‐fold increase in protein‐specific activity were achieved. Hence, partial application of oxygen‐related stress in the two‐compartment system was proposed as a process technology to enhance protein production.     

Split-enzyme fragment as a single affinity tag that enables protein expression,purification, and functional assays

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.     

Secretion of the antibacterial recombinant protein enbocin

The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant enbocin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins.     

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.     

Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system

Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.     

Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells

As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescence intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development.     

Avidin is a promising tag for fusion proteins produced in baculovirus-infected insect cells.

The baculovirus expression vector system (BEVS) has become one of the most versatile and powerful eukaryotic systems for recombinant protein expression. We have constructed a novel baculovirus transfer vector (pbacAVs+C) which allows for the efficient production, detection, and single-step purification of the desired molecule as a secretion-compatible avidin fusion protein in insect cells. It also enables fast construction of the baculoviruses by site-specific transposition in Escherichia coli. To demonstrate the power of this vector, we report here on the production of immunologically intact hevein, a major cysteine-rich latex allergen, as avidin fusion protein. Our results indicate that avidin is a stable and versatile tag in the BEVS. It retains its extraordinarily high biotin-binding activity and also enables independent folding of the fusion partner. The versatility with which avidin fusion proteins can be detected, purified, and immobilized is the basis for the use of our system as a useful alternative in eukaryotic fusion protein production.