scholarly journals Cytoskeleton members, MVBs and the ESCRT-III HvSNF7s are putative key players for protein sorting into protein bodies during barley endosperm development

2019 ◽  
Author(s):  
Valentin Roustan ◽  
Julia Hilscher ◽  
Marieluise Weidinger ◽  
Siegfried Reipert ◽  
Azita Shabrangy ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Endosperm Development ◽  
Protein Bodies ◽  
Grain Development ◽  
Endomembrane System ◽  
Development Stages ◽  
Barley Endosperm ◽  
High Yields

AbstractCereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. PBs can be used as efficient biotechnological systems to produce high yields of stable recombinant proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality, yield and recombinant protein production. Barley, a cereal crop of worldwide importance for the brewing industry, animal feed and to a lesser extent, human nutrition, has been identified as promising candidate for recombinant protein production. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By usingin vivolabel-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 95 proteins associated with the endomembrane system were identified, and 83 of these exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development stages; notably, integration of proteomics data within situsubcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified protein bodies at the early development stages. Endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with protein bodies (PBs) during barley endosperm development. Taken together, our proteomics results specifically identified members of the cytoskeleton, MVBs, and ESCRT as putative key players for protein sorting into PBs during barley endosperm development. These results present a comprehensive overview of proteins involved in the rearrangement of the endomembrane system during barley early grain development and will provide the basis for future work on engineering the endomembrane system to optimize nutrient content and to produce high yields of recombinant proteins.

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

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.

scholarly journals The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

2018 ◽  
Vol 5 (4) ◽  
pp. 89 ◽  
Author(s):  
Albert Uhoraningoga ◽  
Gemma Kinsella ◽  
Gary Henehan ◽  
Barry Ryan
Keyword(s):  
Recombinant Protein ◽  
Design Of Experiments ◽  
Prokaryotic Expression ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Culture Conditions ◽  
Media Composition ◽  
High Yields ◽  
Soluble Recombinant Protein

The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem, statistical models such as Design of Experiments (DoE) have been used to optimise recombinant protein production. This review examines the application of DoE in the production of recombinant proteins in prokaryotic expression systems with specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation designs. It provides examples of DoE applied to optimisation of media and culture conditions.

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

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.

scholarly journals Bacterial signal peptides: structure, optimization, and applications

Eureka ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Esra Erkut
Keyword(s):  
Recombinant Proteins ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Bacterial Species ◽  
Structure Optimization ◽  
Biochemical Properties ◽  
Signal Peptides ◽  
Signal Sequences ◽  
Transport Pathways ◽  
E Coli

Bacterial signal peptides are N-terminal tags that direct proteins for export through one of various transport pathways. These signal peptides are highly important as they are the key determinants of transport, ensuring that the correct protein ends up at the correct pathway. While these peptides consist of three domains with well conserved biochemical properties, there still remains a large amount of diversity between the signal sequences for different proteins, transport pathways, and bacterial species. Recent advancements have allowed us to predict signal sequences and manipulate them in an attempt to optimize export efficiency. This knowledge can then be exploited in the field of recombinant protein production wherein bacterial species can be used to produce and secrete proteins of interest. By fusing the protein with an optimized signal peptide, the yield or rate of export can be improved. This review focuses on signal peptides for two primary transport pathways (Sec and Tat) in E. coli specifically, with an emphasis on applications and the production of recombinant proteins.

scholarly journals Protein sorting into protein bodies during barley endosperm development is putatively regulated by cytoskeleton members, MVBs and the HvSNF7s

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Valentin Roustan ◽  
Julia Hilscher ◽  
Marieluise Weidinger ◽  
Siegfried Reipert ◽  
Azita Shabrangy ◽  
...  
Keyword(s):  
Protein Sorting ◽  
Endosperm Development ◽  
Protein Bodies ◽  
Barley Endosperm

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

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.

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

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.

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

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
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.

scholarly journals Recombinant Protein Production byIn VivoPolymer Inclusion Display

2011 ◽  
Vol 77 (18) ◽  
pp. 6706-6709 ◽  
Author(s):  
Katrin Grage ◽  
Verena Peters ◽  
Bernd H. A. Rehm
Keyword(s):  
Recombinant Protein ◽  
Fusion Protein ◽  
Recombinant Proteins ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Target Protein ◽  
Pha Synthase ◽  
Novel Approach ◽  
Pure Target

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.

scholarly journals Gene Delivery into Plant Cells for Recombinant Protein Production

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
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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