Methods for Efficient High-Throughput Screening of Protein Expression in Recombinant Pichia pastoris Strains

2014 ◽  
pp. 113-123 ◽  
Author(s):  
Andrea Camattari ◽  
Katrin Weinhandl ◽  
Rama K. Gudiminchi
Keyword(s):  
Pichia Pastoris ◽  
Protein Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Recombinant Pichia Pastoris

An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains

2012 ◽  
Vol 19 (11) ◽  
pp. 3046-3054 ◽  
Author(s):  
Li Shen ◽  
Xiao-liang Wang ◽  
Jia Zheng ◽  
Xiao Wang ◽  
Qin-hua Chen ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
High Throughput ◽  
High Throughput Screening ◽  
Plate Assay ◽  
Recombinant Pichia Pastoris

High throughput screening identifies novel, cell cycle-arresting small molecule enhancers of transient protein expression

2017 ◽  
Vol 33 (6) ◽  
pp. 1579-1588 ◽  
Author(s):  
Hermann-Josef Meyer ◽  
Rebecca Turincio ◽  
Shirley Ng ◽  
Juan Li ◽  
Blair Wilson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transient Protein Expression ◽  
Small Molecule Enhancers

scholarly journals High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

2020 ◽  
Author(s):  
Jicong Cao ◽  
Eva Maria Novoa ◽  
Zhizhuo Zhang ◽  
William C.W. Chen ◽  
Dianbo Liu ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Viral Gene ◽  
Cmv Promoter ◽  
Position Effects ◽  
Gene Therapies ◽  
Integration Strategy ◽  
Combinatorial Assembly ◽  
High Throughput Screens

ABSTRACTDespite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5’ untranslated regions (5’ UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5’UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5’ UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5’ UTRs. A total of ∼12,000 5’ UTRs were then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identified three synthetic 5’ UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5’ UTRs enabled even higher protein expression than obtained with each individual 5’ UTR. In summary, we demonstrate that high-throughput screening of 5’ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

scholarly journals Fluorescent probe for high-throughput screening of membrane protein expression

2013 ◽  
Vol 22 (8) ◽  
pp. 1124-1132 ◽  
Author(s):  
A. E. Backmark ◽  
N. Olivier ◽  
A. Snijder ◽  
E. Gordon ◽  
N. Dekker ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Expression ◽  
Fluorescent Probe ◽  
High Throughput ◽  
High Throughput Screening ◽  
Membrane Protein Expression

scholarly journals High-throughput 5′ UTR engineering for enhanced protein production in non-viral gene therapies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jicong Cao ◽  
Eva Maria Novoa ◽  
Zhizhuo Zhang ◽  
William C. W. Chen ◽  
Dianbo Liu ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Viral Gene ◽  
Cmv Promoter ◽  
Position Effects ◽  
Gene Therapies ◽  
Integration Strategy ◽  
Viral Gene Therapy ◽  
High Throughput Screens

AbstractDespite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5′ UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5′ UTRs. A total of ~12,000 5′ UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5′ UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5′ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

Gel microdroplet–based high-throughput screening for directed evolution of xylanase-producing Pichia pastoris

2019 ◽  
Vol 128 (6) ◽  
pp. 662-668 ◽  
Author(s):  
Chunxuan Ma ◽  
Zheng Lin Tan ◽  
Ying Lin ◽  
Shuangyan Han ◽  
Xinhui Xing ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals High-Throughput Screening Identifies Two Novel Small Molecule Enhancers of Recombinant Protein Expression

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 353
Author(s):  
Jiasong Chang ◽  
Xiaoxu Chen ◽  
Ruolin Wang ◽  
Run Shi ◽  
Xiaogang Wang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Small Molecules ◽  
Small Molecule ◽  
Recombinant Proteins ◽  
High Throughput ◽  
High Throughput Screening ◽  
Histone Deacetylase Inhibitors ◽  
Recombinant Protein Expression ◽  
Expression Levels

As a primary strategy for production of biological drugs, recombinant proteins produced by transient transfection of mammalian cells are essential for both basic research and industrial production. Here, we established a high-throughput screening platform for improving the expression levels of recombinant proteins. In total, 10,011 small molecule compounds were screened through our platform. After two rounds of screening, we identified two compounds, Apicidin and M-344, that significantly enhanced recombinant protein expression. Both of the selected compounds were histone deacetylase inhibitors, suggesting that the two small molecules increased the expression levels of recombinant proteins by promoting histone acetylation. Moreover, both molecules showed low cytotoxicity. Therefore, our findings suggest that these small molecules may have wide applications in the future.

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