Efficient Production Of Recombinant Proteins Research Articles

Mammalian perfusion cultivation at high L-Arginine concentration for efficient production of recombinant protein by increasing perfusion filter transmission

Cultivations of Chinese Hamster Ovary (CHO) cells in a perfusion setup were conducted in the presence of super physiological concentrations of L-Arginine to investigate the impact on transmission through the perfusion filter for production of a recombinant domain antibody. Our study revealed that the presence of L-Arginine within the range of 30–50 mM had a positive impact on transmission. However, the higher concentrations were found to have a negative correlation with cell viability, and an optimal concentration of approximately 40 mM was identified. The supplementation of L-Arginine improved overall cultivation performance and enhanced product quality attributes. As a result, our findings demonstrate that the supplementation of L-Arginine to mammalian perfusion cultivations stands as an effective method to address transmission issues, exerting a broad impact on process and production of recombinant proteins.

Application and research progress of MultiBac: A review.

Although the traditional Escherichia coli expression system has matured and is cost-effective, the posttranslation modifications of proteins expressed in eukaryotic cells differ significantly from those expressed in E coli. Insect cells have gradually entered the realm of researchers; however, the proteins synthesized by insect cells are somewhat different from those of mammals in terms of modification. Herein, we have introduced a relatively new method. MultiBac, We introduce the development process, characteristics, and applications of MultiBac technology. And provide new methods for basic researchers. MultiBac has evolved into an indispensable tool in the fields of biotechnology and pharmaceuticals, facilitating the efficient production of recombinant proteins and the study of complex protein complexes. Furthermore, its development has benefited from the integration of synthetic biology techniques, providing additional versatility. But it also has some disadvantages. MultiBac technology is poised to become a key tool in unlocking the mysteries of the protein world, propelling the life sciences ever forward. But researchers should consider its limitations when selecting the most appropriate expression system for their specific needs.

Tailored UPRE2 variants for dynamic gene regulation in yeast

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

A low-cost and eco-friendly recombinant protein expression system using copper-containing industrial wastewater.

The development of innovative methods for highly efficient production of recombinant proteins remains a prominent focus of research in the biotechnology field, primarily due to the fact that current commercial protein expression systems rely on expensive chemical inducers, such as isopropyl β-D-thiogalactoside (IPTG). In our study, we designed a novel approach for protein expression by creating a plasmid that responds to copper. This specialized plasmid was engineered through the fusion of a copper-sensing element with an optimized multiple cloning site (MCS) sequence. This MCS sequence can be easily customized by inserting the coding sequences of target recombinant proteins. Once the plasmid was generated, it was introduced into an engineered Escherichia coli strain lacking copA and cueO. With this modified E. coli strain, we demonstrated that the presence of copper ions can efficiently trigger the induction of recombinant protein expression, resulting in the production of active proteins. Most importantly, this expression system can directly utilize copper-containing industrial wastewater as an inducer for protein expression while simultaneously removing copper from the wastewater. Thus, this study provides a low-cost and eco-friendly strategy for the large-scale recombinant protein production. To the best of our knowledge, this is the first report on the induction of recombinant proteins using industrial wastewater.

Construction of an Escherichia coli chassis for efficient biosynthesis of human-like N-linked glycoproteins.

The production of N-linked glycoproteins in genetically engineered Escherichia coli holds significant potential for reducing costs, streamlining bioprocesses, and enhancing customization. However, the construction of a stable and low-cost microbial cell factory for the efficient production of humanized N-glycosylated recombinant proteins remains a formidable challenge. In this study, we developed a glyco-engineered E. coli chassis to produce N-glycosylated proteins with the human-like glycan Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, containing the human glycoform Gal-β-1,4-GlcNAc-β-1,3-. Our initial efforts were to replace various loci in the genome of the E. coli XL1-Blue strain with oligosaccharyltransferase PglB and the glycosyltransferases LsgCDEF to construct the E. coli chassis. In addition, we systematically optimized the promoter regions in the genome to regulate transcription levels. Subsequently, utilizing a plasmid carrying the target protein, we have successfully obtained N-glycosylated proteins with 100% tetrasaccharide modification at a yield of approximately 320mg/L. Furthermore, we constructed the metabolic pathway for sialylation using a plasmid containing a dual-expression cassette of the target protein and CMP-sialic acid synthesis in the tetrasaccharide chassis cell, resulting in a 40% efficiency of terminal α-2,3- sialylation and a production of 65mg/L of homogeneously sialylated glycoproteins in flasks. Our findings pave the way for further exploration of producing different linkages (α-2,3/α-2,6/α-2,8) of sialylated human-like N-glycoproteins in the periplasm of the plug-and-play E. coli chassis, laying a strong foundation for industrial-scale production.

Essential factors, advanced strategies, challenges, and approaches involved for efficient expression of recombinant proteins in Escherichia coli.

Producing recombinant proteins is a major accomplishment of biotechnology in the past century. Heterologous hosts, either eukaryotic or prokaryotic, are used for the production of these proteins. The utilization of microbial host systems continues to dominate as the most efficient and affordable method for biotherapeutics and food industry productions. Hence, it is crucial to analyze the limitations and advantages of microbial hosts to enhance the efficient production of recombinant proteins on a large scale. E. coli is widely used as a host for the production of recombinant proteins. Researchers have identified certain obstacles with this host, and given the growing demand for recombinant protein production, there is an immediate requirement to enhance this host. The following review discusses the elements contributing to the manifestation of recombinant protein. Subsequently, it sheds light on innovative approaches aimed at improving the expression of recombinant protein. Lastly, it delves into the obstacles and optimization methods associated with translation, mentioning both cis-optimization and trans-optimization, producing soluble recombinant protein, and engineering the metal ion transportation. In this context, a comprehensive description of the distinct features will be provided, and this knowledge could potentially enhance the expression of recombinant proteins in E. coli.

Additivities for Soluble Recombinant Protein Expression in Cytoplasm of Escherichia coli

Recombinant protein expression in Escherichia coli is a fundamental technique in molecular biology and biotechnology. This review provides a comprehensive overview of various additivities to enhance the expression levels of soluble recombinant proteins in E. coli. The discussion encompasses five key aspects. Inducer Optimization: strategies for optimizing the inducer concentration to enhance protein expression. Autoinduction system optimization: the examination of glucose, lactose, and glycerol optimization within autoinduction systems to improve protein production. Osmolytes and osmoprotectants: an analysis of the use of osmolytes and osmoprotectants, such as sorbitol and glycine-betaine, to overcome with ease osmotic stress and enhance protein solubility. Ethanol additives: the impact of ethanol on E. coli physiology and its potential to improve recombinant protein expression. Cofactors and metabolic precursors: insights into the addition of cofactors, such as pyridoxal phosphate, riboflavin, thiamine, and pyridoxine, and the utilization of metabolic precursors to enhance the corresponding protein expression. This review highlights both the successful strategies and challenges in recombinant protein expression and provides insights into potential future research directions. Understanding and optimizing these factors is crucial for the efficient production of recombinant proteins for various applications in biotechnology. Furthermore, based on the analyzed data, we propose a straightforward scheme to optimize the additives in the cultivation medium.

Enhanced production of difficult-to-express proteins through knocking down rnpA gene expression.

Escherichia coli has been employed as a workhorse for the efficient production of recombinant proteins. However, some proteins were found to be difficult to produce in E. coli. The stability of mRNA has been considered as one of the important factors affecting recombinant protein production. Here we report a generally applicable and simple strategy for enhancing mRNA stability, and consequently improving recombinant protein production in E. coli. RNase P, a ribozyme comprising an RNA subunit (RnpB) and a protein subunit (RnpA), is involved in tRNA maturation. Based on the finding that purified RnpA can digest rRNA and mRNA in vitro, it was reasoned that knocking down the level of RnpA might enhance recombinant protein production. For this, the synthetic small regulatory RNA-based knockdown system was applied to reduce the expression level of RnpA. The developed RnpA knockdown system allowed successful overexpression of 23 different recombinant proteins of various origins and sizes, including Cas9 protein, antibody fragment, and spider silk protein. Notably, a 284.9-kDa ultra-high molecular weight, highly repetitive glycine-rich spider silk protein, which is one of the most difficult proteins to produce, could be produced to 1.38gL-1 , about two-fold higher than the highest value previously achieved, by a fed-batch culture of recombinant E. coli strain employing the RnpA knockdown system. The RnpA-knockdown strategy reported here will be generally useful for the production of recombinant proteins including those that have been difficult to produce.

Efficient production of recombinant proteins in suspension CHO cells culture using the Tol2 transposon system coupled with cycloheximide resistance selection

DNA recombination techniques in mammalian cells has been applied to the production of therapeutic proteins for several decades. To be used for commercial production, established cell lines should stably express target proteins with high productivity and acceptable quality for human use. In the conventional transfection method, the screening process is laborious and time-consuming since superior cell lines had to be selected from an enormous number of transfected cell pools and clonal cell lines with a wide variety of transgene insertion locations. In this study, we demonstrated that the combination of a Tol2 transposon system and cell selection by cycloheximide resistance is an efficient method to express therapeutic proteins, such as human antibody in suspension culture of Chinese hamster ovary cells. The resulting stable cell lines showed constant productivity and cell growth over a long enough cultivation periods for recombinant protein production. We anticipate that this approach will prove widely applicable to protein production in research and development of pharmaceutical products.

Development of stable HEK293T cell pools expressing CSFV E2 protein: A potential antigen expression platform.

Large quantities of antigens are required since protective antigens, such as classical swine fever virus (CSFV) E2 protein, are widely used in diagnostic reagents and subunit vaccines. Compared to clonal cell lines and transient gene expression, stable cell pools provide a potential alternative platform to rapidly produce large amounts of antigens. In this work, firstly, Human embryonic kidney 293T (HEK293T) cell pools expressing E2 protein were developed by transduction of lentiviral vectors. On the one hand, the SP7 was selected from 7 well-performing signal peptides to remarkably increase the production of E2 protein. On the other hand, it was found that high MOI could improve the expression of E2 protein by increasing gene copy numbers. Moreover, the HEK293T cell pools were evaluated for stability by passages and batch cultures, demonstrating that the cell pools were stable for at least 90days. And then, the performance of the cell pools in batch, fed-batch, and semi-perfusion was studied. Among them, the titer of E2 protein was up to 2g/L in semi-perfusion, which is currently the highest to the authors' knowledge. Finally, the aggregations and immunogenicity of the E2 protein were analyzed by SDS-PAGE and immunization of mice, respectively. There was no significant difference in aggregations and antibody titers of E2 protein in three culture methods. These results suggest that stable HEK293T cell pools are a promising and robust platform for rapid and efficient production of recombinant proteins.

Tagging Recombinant Proteins to Enhance Solubility and Aid Purification.

Protein fusion technology has had a major impact on the efficient production and purification of individual recombinant proteins. The use of genetically engineered affinity and solubility-enhancing polypeptide "tags" has a long history, and there is a considerable repertoire of these that can be used to address issues related to the expression, stability, solubility, folding, and purification of their fusion partner. In the case of large-scale proteomic studies, the development of purification procedures tailored to individual proteins is not practicable, and affinity tags have become indispensable tools for structural and functional proteomic initiatives that involve the expression of many proteins in parallel. In this chapter, the rationale and applications of a range of established and more recently developed solubility-enhancing and affinity tags is described.

Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli: Dissecting and Mitigating Redox Heterogeneity

Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG1-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG1 CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.

The production of recombinant platelet-derived growth factor D using the second generation modified vaccinia Ankara viral system.

The vaccinia virus expression system is known for the efficient production of recombinant proteins with “appropriate” posttranslational modification using desired mammalian cell lines. However, being a replication competent virus, vaccinia virus poses a health threat to immunocompromised individuals and requires biosafety level 2 (BSL2) laboratory precautions, thereby restricting its use by the scientific community. Development of the host range restricted modified vaccinia Ankara (MVA) system has allowed researchers to work with a safer virus even at BSL1. Here, we report on the use of an improved second generation MVA viral system incorporating two selective markers and fluorescent proteins for easier recombinant virus identification. Notably, we demonstrate that this novel system is capable of producing secreted recombinant proteins, a finding not previously reported. Through purification and characterization of wild type and mutant platelet‐derived growth factor D (PDGF D) dimer species, we demonstrate this system is capable of producing the latent full‐length PDGF D dimer, partially processed intermediate dimer (hemidimer), as well as fully processed growth factor domain dimer that show chemical integrity and biological activity. Importantly, this system is amenable to scaling up for the mass production of recombinant PDGF D (rPDGF D) dimer species.

Production of autolysis-proof Kex2 protease from Candida albicans in Saccharomyces cerevisiae for in vitro processing of fusion proteins.

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.

Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells.

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

Improvement of recombinant miraculin production in transgenic tomato by crossbreeding-based genetic background modification.

An important optimization step in plant-based recombinant protein production systems is the selection of an appropriate cultivar after a potential host has been determined. Previously, we have shown that transgenic tomatoes of the variety 'Micro-Tom' accumulate incredibly high levels of miraculin (MIR) due to the introduction of MIR gene controlled by a CaMV35S promoter and a heat-shock protein terminator. However, 'Micro-Tom' is unsuitable for commercial production of MIR as it is a dwarf cultivar characterized by small-sized fruit and poor yield. Here, we used the crossbreeding approach to transfer the high MIR accumulation trait of transgenic 'Micro-Tom' tomatoes to 'Natsunokoma' and 'Aichi First', two commercial cultivars producing medium and large fruit sizes, respectively. Fruits of the resultant crossbred lines were larger (~ 95 times), but their miraculin accumulation levels (~ 1,062μg/g fresh mass) were comparable to the donor cultivar, indicating that the high miraculin accumulation trait was preserved regardless of fruit size or cultivar. Further, the transferred trait resulted in a 3-4fold increase in overall miraculin production than that of the previously reported line 5B. These findings demonstrate the effectiveness of crossbreeding in improving MIR production in tomatoes and could pave the way for a more efficient production of recombinant proteins in other plants.

Expanding the Scope of Genetically Encoded Lysine Post-Translational Modifications with Lactylation, β-Hydroxybutyrylation and Lipoylation.

Post-translational modifications (PTMs) occurring on lysine residues, especially diverse forms of acylations, have seen rapid growth over the past two decades. Among them, lactylation and β-hydroxybutyrylation of lysine side-chains are newly identified histone marks and their implications in physiology and diseases have aroused broad research interest. Meanwhile, lysine lipoylation is highly conserved in diverse organisms and well known for its pivotal role in central metabolic pathways. Recent findings in the proteomic profiling of protein lipoylation have nonetheless suggested a pressing need for an extensive investigation. For both basic and applied research, it is necessary to prepare PTM-bearing proteins particularly in a site-specific manner. Herein, we use genetic code expansion to site-specifically generate these lysine PTMs, including lactylation, β-hydroxybutyrylation and lipoylation in proteins in E. coli and mammalian cells. Notably, using strategies including activity-based selection, screening and rational design, unique pyrrolysyl-tRNA synthetase variants were successfully evolved for each of the three non-canonical amino acids, which enabled efficient production of recombinant proteins. Through encoding these ncAAs, we examined the deacylase activities of mammalian sirtuins to these modifications, and importantly we unfold the lipoamidase activity of several sirtuins.

Developing a secretory AcGFP1-based IRES expression system for efficient production of mammalian recombinant proteins

To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening.

Production of soluble regulatory hydrogenase from Ralstonia eutropha in Escherichia coli using a fed-batch-based autoinduction system

BackgroundAutoinduction systems can regulate protein production in Escherichia coli without the need to monitor cell growth or add inducer at the proper time following culture growth. Compared to classical IPTG induction, autoinduction provides a simple and fast way to obtain high protein yields. In the present study, we report on the optimization process for the enhanced heterologous production of the Ralstonia eutropha regulatory hydrogenase (RH) in E. coli using autoinduction. These autoinduction methods were combined with the EnPresso B fed-batch like growth system, which applies slow in situ enzymatic glucose release from a polymer to control cell growth and protein synthesis rate.ResultsWe were able to produce 125 mg L−1 RH corresponding to a productivity averaged over the whole process time of 3 mg (L h)−1 in shake flasks using classic single-shot IPTG induction. IPTG autoinduction resulted in a comparable volumetric RH yield of 112 mg L−1 and due to the shorter overall process time in a 1.6-fold higher productivity of 5 mg (L h)−1. In contrast, lactose autoinduction increased the volumetric yield more than 2.5-fold and the space time yield fourfold reaching 280 mg L−1 and 11.5 mg (L h)−1, respectively. Furthermore, repeated addition of booster increased RH production to 370 mg L−1, which to our knowledge is the highest RH concentration produced in E. coli to date.ConclusionsThe findings of this study confirm the general feasibility of the developed fed-batch based autoinduction system and provide an alternative to conventional induction systems for efficient recombinant protein production. We believe that the fed-batch based autoinduction system developed herein will favor the heterologous production of larger quantities of difficult-to-express complex enzymes to enable economical production of these kinds of proteins.

Efficient Transient Expression of Recombinant Proteins Using DNA Viral Vectors in Freshwater Microalgal Species.

The increase in the world population, the advent of new infections and health issues, and the scarcity of natural biological products have spotlighted the importance of recombinant protein technology and its large-scale production in a cost-effective manner. Microalgae have become a significant promising platform with the potential to meet the increasing demand for recombinant proteins and other biologicals. Microalgae are safe organisms that can grow rapidly and are easily cultivated with basic nutrient requirements. Although continuous efforts have led to considerable progress in the algae genetic engineering field, there are still many hurdles to overcome before these microorganisms emerge as a mature expression system. Hence, there is a need to develop efficient expression approaches to exploit microalgae for the production of recombinant proteins at convenient yields. This study aimed to test the ability of the DNA geminiviral vector with Rep-mediated replication to transiently express recombinant proteins in the freshwater microalgal species Chlamydomonas reinhardtii and Chlorella vulgaris using Agrobacterium-mediated transformation. The SARS-CoV-2 receptor binding domain (RBD) and basic fibroblast growth factor (bFGF) are representative antigen proteins and growth factor proteins, respectively, that were subcloned in a geminiviral vector and were used for nuclear transformation to transiently express these proteins in C. reinhardtii and C. vulgaris. The results showed that the geminiviral vector allowed the expression of both recombinant proteins in both algal species, with yields at 48 h posttransformation of up to 1.14 μg/g RBD and 1.61 ng/g FGF in C. vulgaris and 1.61 μg/g RBD and 1.025 ng/g FGF in C. reinhardtii. Thus, this study provides a proof of concept for the use of DNA viral vectors for the simple, rapid, and efficient production of recombinant proteins that repress the difficulties faced in the genetic transformation of these unicellular green microalgae. This concept opens an avenue to explore and optimize green microalgae as an ideal economically valuable platform for the production of therapeutic and industrially relevant recombinant proteins in shorter time periods with significant yields.