Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants

2015 ◽  
Vol 10 (9) ◽  
pp. 1478-1486 ◽  
Author(s):  
Philippe V. Jutras ◽  
Marc-André D'Aoust ◽  
Manon M.-J. Couture ◽  
Louis-Philippe Vézina ◽  
Marie-Claire Goulet ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Stability ◽  
Secretory Pathway ◽  
Proton Channel

scholarly journals Recombinant Protein Stability in Cyanobacteria

2021 ◽  
Vol 10 (4) ◽  
pp. 810-825
Author(s):  
Xianan Zhang ◽  
Nico Betterle ◽  
Diego Hidalgo Martinez ◽  
Anastasios Melis
Keyword(s):  
Recombinant Protein ◽  
Protein Stability

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

2011 ◽  
Vol 78 (1) ◽  
pp. 38-47 ◽  
Author(s):  
Ariane Leites Larentis ◽  
Ana Paula Corrêa Argondizzo ◽  
Gabriela dos Santos Esteves ◽  
Ellen Jessouron ◽  
Ricardo Galler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Stability ◽  
Expression In Escherichia Coli ◽  
Long Term Storage ◽  
Term Storage

scholarly journals Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins

2021 ◽  
Author(s):  
Magdalena Malm ◽  
Chih-Chung Kuo ◽  
Mona Moradi Barzadd ◽  
Aman Mebrahtu ◽  
Num Wistabacka ◽  
...  
Keyword(s):  
Systems Biology ◽  
Recombinant Protein ◽  
Host Cell ◽  
Secretory Pathway ◽  
Protein Yield ◽  
Expression Levels ◽  
Expression Variation ◽  
Human Proteins ◽  
Significant Expression

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.

Characterization of VIP36, an animal lectin homologous to leguminous lectins

1996 ◽  
Vol 109 (1) ◽  
pp. 271-276 ◽  
Author(s):  
K. Fiedler ◽  
K. Simons
Keyword(s):  
Golgi Apparatus ◽  
Recombinant Protein ◽  
Secretory Pathway ◽  
Mdck Cells ◽  
Membrane Structures ◽  
New Family ◽  
Legume Lectin ◽  
Animal Lectin

VIP36 was isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes. The protein is localized to the Golgi apparatus and the cell surface, and belongs to a new family of legume lectin homologues in the animal secretory pathway that might be involved in the trafficking of glycoproteins, glycolipids or both. Here we show that VIP36 is N-glycosylated and expressed in organs abundant in epithelial cells as well as in non-epithelial organs. Our studies demonstrate that the recombinant exoplasmic/luminal domain of VIP36 binds Ca2+ and that the protein decorates internal membrane structures of MDCK cells in vitro that are distinct from the Golgi apparatus. This binding requires Ca2+ and can be specifically inhibited by N-acetyl-D-galactosamine. The recombinant protein was used for affinity chromatography. Glycopeptides obtained from [3H]galactose-labelled cells bind to VIP36 and can be eluted with N-acetyl-D-galactosamine. Our data imply that VIP36 functions as a lectin in post-Golgi trafficking.

scholarly journals Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production

2018 ◽  
Vol 115 (47) ◽  
pp. E11025-E11032 ◽  
Author(s):  
Mingtao Huang ◽  
Guokun Wang ◽  
Jiufu Qin ◽  
Dina Petranovic ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Protein ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Pathway ◽  
Synergistic Effects ◽  
Production Capacity ◽  
Market Demand ◽  
Protein Retention ◽  
Cell Factories

Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.

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 In situ detection of protein interactions for recombinant therapeutic enzymes

2020 ◽  
Author(s):  
Mojtaba Samoudi ◽  
Chih-Chung Kuo ◽  
Caressa M. Robinson ◽  
Km Shams-Ud-Doha ◽  
Song-Min Schinn ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Ppi Networks

AbstractDespite their therapeutic potential, many protein drugs remain inaccessible to patients since they are difficult to secrete. Each recombinant protein has unique physicochemical properties and requires different machinery for proper folding, assembly, and post-translational modifications (PTMs). Here we aimed to identify the machinery supporting recombinant protein secretion by measuring the protein-protein interaction (PPI) networks of four different recombinant proteins (SERPINA1, SERPINC1, SERPING1 and SeAP) with various PTMs and structural motifs using the proximity-dependent biotin identification (BioID) method. We identified PPIs associated with specific features of the secreted proteins using a Bayesian statistical model, and found proteins involved in protein folding, disulfide bond formation and N-glycosylation were positively correlated with the corresponding features of the four model proteins. Among others, oxidative folding enzymes showed the strongest association with disulfide bond formation, supporting their critical roles in proper folding and maintaining the ER stability. Knockdown of disulfide-isomerase PDIA4, a measured interactor with significance for SERPINC1 but not SERPINA1, led to the decreased secretion of SERPINC1, which relies on its extensive disulfide bonds, compared to SERPINA1, which has no disulfide bonds. Proximity-dependent labeling successfully identified the transient interactions supporting synthesis of secreted recombinant proteins and refined our understanding of key molecular mechanisms of the secretory pathway during recombinant protein production.

scholarly journals Exploiting Prophage-Mediated Lysis for Biotherapeutic Release byLactobacillus reuteri

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Laura M. Alexander ◽  
Jee-Hwan Oh ◽  
Donald S. Stapleton ◽  
Kathryn L. Schueler ◽  
Mark P. Keller ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Secretory Pathway ◽  
Lactobacillus Reuteri ◽  
Gastrointestinal Transit ◽  
Biologically Active ◽  
Content Type ◽  
Extracellular Milieu ◽  
Therapeutic Molecules

ABSTRACTLactobacillus reuterihas the potential to be developed as a microbial therapeutic delivery platform because of an established safety profile, health-promoting properties, and available genome editing tools. Here, we show thatL. reuteriVPL1014 exhibits a low mutation rate compared to other Gram-positive bacteria, which we expect will contribute to the stability of genetically modified strains. VPL1014 encodes two biologically active prophages, which are induced during gastrointestinal transit. We hypothesized that intracellularly accumulated recombinant protein can be released following bacteriophage-mediated lysis. To test this, we engineered VPL1014 to accumulate leptin, our model protein, inside the cell.In vitroprophage induction of recombinant VPL1014 released leptin into the extracellular milieu, which corresponded to bacteriophage production. We also employed a plasmid system that does not require antibiotic in the growth medium for plasmid maintenance. Collectively, these data provide new avenues to exploit native prophages to deliver therapeutic molecules.IMPORTANCELactic acid bacteria (LAB) have been explored as potential biotherapeutic vehicles for the past 20 years. To secrete a therapeutic in the extracellular milieu, one typically relies on the bacterial secretion pathway, i.e., the Sec pathway. Overexpression of a secreted protein can overload the secretory pathway and impact the organism’s fitness, and optimization of the signal peptide is also required to maximize the efficiency of the release of mature protein. Here, we describe a previously unexplored approach to release therapeutics from the probioticLactobacillus reuteri. We demonstrate that an intracellularly accumulated recombinant protein is released following prophage activation. Since we recently demonstrated that prophages are activated during gastrointestinal transit, we propose that this method will provide a straightforward and efficient approach to deliver therapeuticsin vivo.

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