Recombinant Protein Production byIn VivoPolymer Inclusion Display

ABSTRACTA novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion.

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Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

Frontiers in Microbiology ◽

10.3389/fmicb.2021.682001 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gema Lozano Terol ◽

Julia Gallego-Jara ◽

Rosa Alba Sola Martínez ◽

Adrián Martínez Vivancos ◽

Manuel Cánovas Díaz ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Proteins ◽

Copy Number ◽

Recombinant Protein Production ◽

Protein Production ◽

Recombinant Protein Expression ◽

Expression System ◽

E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

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CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain

Frontiers in Plant Science ◽

10.3389/fpls.2021.755788 ◽

2021 ◽

Vol 12 ◽

Author(s):

Michael Panting ◽

Inger Baeksted Holme ◽

Jón Már Björnsson ◽

Yingxin Zhong ◽

Henrik Brinch-Pedersen

Keyword(s):

Recombinant Protein ◽

Er Stress ◽

Unfolded Protein Response ◽

Recombinant Proteins ◽

Recombinant Protein Production ◽

Protein Production ◽

Protein Accumulation ◽

Unfolded Protein ◽

Protein Response ◽

Positive Effect

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

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Genome scale modeling of the protein secretory pathway reveals novel targets for improved recombinant protein production in yeast

10.1101/2021.10.16.464630 ◽

2021 ◽

Author(s):

Feiran Li ◽

Yu Chen ◽

Qi Qi ◽

Yanyan Wang ◽

Le Yuan ◽

...

Keyword(s):

Recombinant Protein ◽

Recombinant Proteins ◽

Recombinant Protein Production ◽

Protein Production ◽

Secretory Pathway ◽

Protein Translation ◽

Cellular Growth ◽

Secretory Proteins ◽

Yeast Saccharomyces Cerevisiae ◽

Genome Scale

Eukaryal cells are used for the production of many recombinant pharmaceutical proteins, including several of the current top-selling products. The protein secretory pathway in eukaryal cells is complex and involves many different processes such as post-translational modifications, translocation, and folding. Furthermore, recombinant protein production competes with native secretory proteins for the limited energy and proteome resources allocated to the protein secretory pathway. Due to the complexity of this pathway, improvement through metabolic engineering has traditionally been relatively ad-hoc; and considering the industrial importance of this pathway, there is a need for more systematic approaches for novel design principles. Here, we present the first proteome-constrained genome-scale protein secretory model of a eukaryal cell, namely for the yeast Saccharomyces cerevisiae (pcSecYeast). The model contains all key processes of this pathway, i.e., protein translation, modification, and degradation coupled with metabolism. The model can capture delicate phenotypic changes such as the switch in the use of specific glucose transporters in response to changing extracellular glucose concentration. Furthermore, the model can also simulate the effects of protein misfolding on cellular growth, suggesting that retro-translocation of misfolded proteins contributes to protein retention in the Endoplasmic reticulum (ER). We used pcSecYeast to simulate various recombinant proteins production and identified overexpression targets for different recombinant proteins overproduction. We experimentally validated many of the predicted targets for α-amylase production in this study, and the results show that the secretory pathways have more limited capacity than metabolism in terms of protein secretion.

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Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites

PLoS Computational Biology ◽

10.1371/journal.pcbi.1009461 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1009461

Author(s):

Bikash K. Bhandari ◽

Chun Shen Lim ◽

Daniela M. Remus ◽

Augustine Chen ◽

Craig van Dolleweerd ◽

...

Keyword(s):

Cell Growth ◽

Recombinant Protein ◽

Recombinant Proteins ◽

Translation Initiation ◽

Recombinant Protein Production ◽

Protein Production ◽

Synonymous Codon ◽

Recombinant Protein Expression ◽

Host Cells ◽

Over Expression

Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann’s ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during overexpression.

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Development of an Autoinducible Plasmid for Recombinant Protein Production

Protein and Peptide Letters ◽

10.2174/0929866528666211105113750 ◽

2021 ◽

Vol 28 ◽

Author(s):

Young Kee Chae ◽

Hakbeom Kim

Keyword(s):

Recombinant Protein ◽

Stationary Phase ◽

Recombinant Protein Production ◽

Protein Production ◽

Binding Protein ◽

Maltose Binding Protein ◽

Target Protein ◽

Expression Vectors ◽

Lethal Gene ◽

E Coli

Background: The production of recombinant proteins in E. coli involves such factors as host strains, expression vectors, culture media, and induction methods. The typical procedure to produce heterologous proteins consists of the following: (1) insertion of the target gene into a suitable vector to construct an overexpression plasmid, (2) transformation of a strain specialized for protein production with the constructed plasmid DNA, (3) growth of the host in a suitable medium and induction of the protein production at a right moment, and (4) further growth to get the maximum yield. There are hurdles involved in each of these steps, and researchers have developed many materials or methods, which often require special recipes or procedures. Objective: To eliminate the special requirements for the recombinant protein production by using readily available materials. Also to save time and effort in the routine protein production work. Method: We started with a vector capable of producing a target protein fused to the C-terminus of the maltose binding protein (MBP). The mCherry (red fluorescent protein) gene was fused to MBP. It acted as a reporter in the initial screening procedure. The original lethal gene (barnase) was replaced with sacB. We chose 3 stationary phase promoters, and made hybrids of them by mixing halves from each one. The T5 promoter was replaced with these stationary phase promoters or their hybrids. The best plasmid was selected by the color intensity of the cell pellet. MBP and GST genes were inserted in place of sacB, and their production yields were compared with the original plasmid in the conventional way of expression. Results: We constructed an expression plasmid with an autoinducible promoter working in a host that was not specially designed for protein production and in a TB medium which did not contain any secret ingredient, nor was difficult to prepare unlike Studier’s defined medium. This plasmid also contains a color indicator which turns red when protein production is successful. We tested our system with the maltose binding protein (MBP) and the glutathione S-transferase (GST), and showed that both proteins were produced to a level comparable to what the commercial medium and/or the specialized strain yielded. Conclusion: We developed a plasmid equipped with an autoinducible promoter, a hybrid of the two promoters which were activated at the stationary phase. This plasmid does not need a special E. coli strain nor a sophisticated nor an expensive medium. It produces an intense red (or pink) color, which can be used as an indicator of a successful production of the target protein and as a predictive measure of the amount of the produced target protein. We speculate that this plasmid will have its greatest advantage when growing cells at low temperatures which would inevitably take a long time.

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Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective

BioMed Research International ◽

10.1155/2014/136419 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 64

Author(s):

Matilde Merlin ◽

Elisa Gecchele ◽

Stefano Capaldi ◽

Mario Pezzotti ◽

Linda Avesani

Keyword(s):

Recombinant Protein ◽

Recombinant Proteins ◽

Comparative Evaluation ◽

Recombinant Protein Production ◽

Protein Production ◽

Individual System ◽

Pharmaceutical Proteins ◽

Wide Range

In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms.

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Gene Delivery into Plant Cells for Recombinant Protein Production

BioMed Research International ◽

10.1155/2015/932161 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 17

Author(s):

Qiang Chen ◽

Huafang Lai

Keyword(s):

Gene Delivery ◽

Recombinant Protein ◽

Recombinant Proteins ◽

Recombinant Protein Production ◽

Large Scale ◽

Protein Production ◽

Plant Cells ◽

High Volume ◽

Extensive Discussion ◽

Transgene Delivery

Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration inNicotianaand non-Nicotianaplant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

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