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Protein Engineering Design & Selection最新文献

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Improved thermostability of proteinase K and recognizing the synergistic effect of Rosetta and FoldX approaches. 改善蛋白酶K的热稳定性，并认识到Rosetta和FoldX方法的协同效应。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab024
Yang Zhao, Daixi Li, Xue Bai, Manjie Luo, Yan Feng, Yilei Zhao, Fuqiang Ma, Guang-Yu Yang
{"title":"Improved thermostability of proteinase K and recognizing the synergistic effect of Rosetta and FoldX approaches.","authors":"Yang Zhao,&nbsp;Daixi Li,&nbsp;Xue Bai,&nbsp;Manjie Luo,&nbsp;Yan Feng,&nbsp;Yilei Zhao,&nbsp;Fuqiang Ma,&nbsp;Guang-Yu Yang","doi":"10.1093/protein/gzab024","DOIUrl":"https://doi.org/10.1093/protein/gzab024","url":null,"abstract":"<p><p>Proteinase K (PRK) is a proteolytic enzyme that has been widely used in industrial applications. However, poor stability has severely limited the uses of PRK. In this work, we used two structure-guided rational design methods, Rosetta and FoldX, to modify PRK thermostability. Fifty-two single amino acid conversion mutants were constructed based on software predictions of residues that could affect protein stability. Experimental characterization revealed that 46% (21 mutants) exhibited enhanced thermostability. The top four variants, D260V, T4Y, S216Q, and S219Q, showed improved half-lives at 69°C by 12.4-, 2.6-, 2.3-, and 2.2-fold that of the parent enzyme, respectively. We also found that selecting mutations predicted by both methods could increase the predictive accuracy over that of either method alone, with 73% of the shared predicted mutations resulting in higher thermostability. In addition to providing promising new variants of PRK in industrial applications, our findings also show that combining these programs may synergistically improve their predictive accuracy.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39535240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 4
Breakthroughs in computational design methods open up new frontiers for de novo protein engineering. 计算设计方法的突破为从头开始的蛋白质工程开辟了新的领域。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab007
Ben A Meinen, Christopher D Bahl
{"title":"Breakthroughs in computational design methods open up new frontiers for de novo protein engineering.","authors":"Ben A Meinen,&nbsp;Christopher D Bahl","doi":"10.1093/protein/gzab007","DOIUrl":"https://doi.org/10.1093/protein/gzab007","url":null,"abstract":"<p><p>Proteins catalyze the majority of chemical reactions in organisms, and harnessing this power has long been the focus of the protein engineering field. Computational protein design aims to create new proteins and functions in silico, and in doing so, accelerate the process, reduce costs and enable more sophisticated engineering goals to be accomplished. Challenges that very recently seemed impossible are now within reach thanks to several landmark advances in computational protein design methods. Here, we summarize these new methods, with a particular emphasis on de novo protein design advancements occurring within the past 5 years.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzab007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38896767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
Linking thermodynamics and measurements of protein stability. 把热力学和蛋白质稳定性的测量联系起来。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab002
Kresten Lindorff-Larsen, Kaare Teilum
{"title":"Linking thermodynamics and measurements of protein stability.","authors":"Kresten Lindorff-Larsen,&nbsp;Kaare Teilum","doi":"10.1093/protein/gzab002","DOIUrl":"https://doi.org/10.1093/protein/gzab002","url":null,"abstract":"<p><p>We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding. The primary focus is on the thermodynamics of reversible folding/unfolding transitions and the experimental methods that are available for extracting thermodynamic parameters. We highlight the importance of modelling both how the folding equilibrium depends on a perturbing variable such as temperature or denaturant concentration, and the importance of modelling the baselines in the experimental observables.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzab002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25482572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
Engineering stable carbonic anhydrases for CO2 capture: a critical review. 工程稳定的碳酸酐酶用于二氧化碳捕获:一个重要的回顾。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab021
Mirfath Sultana Mesbahuddin, Aravindhan Ganesan, Subha Kalyaanamoorthy
{"title":"Engineering stable carbonic anhydrases for CO2 capture: a critical review.","authors":"Mirfath Sultana Mesbahuddin,&nbsp;Aravindhan Ganesan,&nbsp;Subha Kalyaanamoorthy","doi":"10.1093/protein/gzab021","DOIUrl":"https://doi.org/10.1093/protein/gzab021","url":null,"abstract":"<p><p>Targeted inhibition of misregulated protein-protein interactions (PPIs) has been a promising area of investigation in drug discovery and development for human diseases. However, many constraints remain, including shallow binding surfaces and dynamic conformation changes upon interaction. A particularly challenging aspect is the undesirable off-target effects caused by inherent structural similarity among the protein families. To tackle this problem, phage display has been used to engineer PPIs for high-specificity binders with improved binding affinity and greatly reduced undesirable interactions with closely related proteins. Although general steps of phage display are standardized, library design is highly variable depending on experimental contexts. Here in this review, we examined recent advances in the structure-based combinatorial library design and the advantages and limitations of different approaches. The strategies described here can be explored for other protein-protein interactions and aid in designing new libraries or improving on previous libraries.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39338822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
A versatile assay platform for enzymatic poly(ethylene-terephthalate) degradation. 一个通用的酶促聚对苯二甲酸乙酯降解测定平台。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab022
Sebastian Weigert, Andreas Gagsteiger, Teresa Menzel, Birte Höcker
{"title":"A versatile assay platform for enzymatic poly(ethylene-terephthalate) degradation.","authors":"Sebastian Weigert,&nbsp;Andreas Gagsteiger,&nbsp;Teresa Menzel,&nbsp;Birte Höcker","doi":"10.1093/protein/gzab022","DOIUrl":"https://doi.org/10.1093/protein/gzab022","url":null,"abstract":"<p><p>Accumulation of plastic and subsequent microplastic is a major environmental challenge. With the discovery of potent polyethylene terephthalate (PET)-degrading enzymes, a new perspective arose for environmental decomposition as well as technical recycling. To explore the enormous diversity of potential PET-degrading enzymes in nature and also to conveniently employ techniques like protein engineering and directed evolution, a fast and reliable assay platform is needed. In this study we present our versatile solution applying a PET coating on standard lab consumables such as polymerase chain reaction tubes, 96- and 384-well microtiter plates, yielding an adjustable crystallinity of the PET. Combining the reaction vessels with either ultra-high performance liquid chromatography (UHPLC) or fluorometric readout and additional enzyme quantification offers a range of advantages. Thereby, the platform can easily be adapted to diverse needs from detailed analysis with high precision to high-throughput (HT) applications including crude lysate analysis.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39338823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Rational engineering of an erythropoietin fusion protein to treat hypoxia. 合理设计促红细胞生成素融合蛋白治疗缺氧。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab025
Jungmin Lee, Andyna Vernet, Nathalie G Gruber, Kasia M Kready, Devin R Burrill, Jeffrey C Way, Pamela A Silver
{"title":"Rational engineering of an erythropoietin fusion protein to treat hypoxia.","authors":"Jungmin Lee,&nbsp;Andyna Vernet,&nbsp;Nathalie G Gruber,&nbsp;Kasia M Kready,&nbsp;Devin R Burrill,&nbsp;Jeffrey C Way,&nbsp;Pamela A Silver","doi":"10.1093/protein/gzab025","DOIUrl":"https://doi.org/10.1093/protein/gzab025","url":null,"abstract":"<p><p>Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39835737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
High-efficacy, high-manufacturability human VH domain antibody therapeutics from transgenic sources. 来自转基因来源的高效、高可制造性人VH结构域抗体疗法。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab012
Kasandra Bélanger, Jamshid Tanha
{"title":"High-efficacy, high-manufacturability human VH domain antibody therapeutics from transgenic sources.","authors":"Kasandra Bélanger,&nbsp;Jamshid Tanha","doi":"10.1093/protein/gzab012","DOIUrl":"https://doi.org/10.1093/protein/gzab012","url":null,"abstract":"<p><p>Interest in single-domain antibodies (sdAbs) stems from their unique structural/pronounced, hence therapeutically desirable, features. From the outset-as therapeutic modalities-human antibody heavy chain variable domains (VHs) attracted a particular attention compared with 'naturally-occurring' camelid and shark heavy-chain-only antibody variable domains (VHHs and VNARs, respectively) due to their perceived lack of immunogenicity. However, they have not quite lived up to their initial promise as the VH hits, primarily mined from synthetic VH phage display libraries, have too often been plagued with aggregation tendencies, low solubility and low affinity. Largely unexplored, synthetic camelized human VH display libraries appeared to have remediated the aggregation problem, but the low affinity of the VH hits still persisted, requiring undertaking additional, laborious affinity maturation steps to render VHs therapeutically feasible. A wholesome resolution has recently emerged with the development of non-canonical transgenic rodent antibody discovery platforms that appear to facilely and profusely generate high affinity, high solubility and aggregation-resistant human VHs.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzab012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38914156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Recent developments in engineering protein-protein interactions using phage display. 利用噬菌体展示技术工程蛋白-蛋白相互作用的最新进展。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab014
Chen T Liang, Olivia M A Roscow, Wei Zhang
{"title":"Recent developments in engineering protein-protein interactions using phage display.","authors":"Chen T Liang,&nbsp;Olivia M A Roscow,&nbsp;Wei Zhang","doi":"10.1093/protein/gzab014","DOIUrl":"https://doi.org/10.1093/protein/gzab014","url":null,"abstract":"<p><p>Targeted inhibition of misregulated protein-protein interactions (PPIs) has been a promising area of investigation in drug discovery and development for human diseases. However, many constraints remain, including shallow binding surfaces and dynamic conformation changes upon interaction. A particularly challenging aspect is the undesirable off-target effects caused by inherent structural similarity among the protein families. To tackle this problem, phage display has been used to engineer PPIs for high-specificity binders with improved binding affinity and greatly reduced undesirable interactions with closely related proteins. Although general steps of phage display are standardized, library design is highly variable depending on experimental contexts. Here in this review, we examined recent advances in the structure-based combinatorial library design and the advantages and limitations of different approaches. The strategies described here can be explored for other protein-protein interactions and aid in designing new libraries or improving on previous libraries.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39085728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region. 在核糖核酸酶H区引入二硫桥改善Moloney小鼠白血病病毒逆转录酶的热稳定性。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab006
Yutaro Narukawa, Mako Kandabashi, Tongyang Li, Misato Baba, Haruka Hara, Kenji Kojima, Kei Iida, Takayoshi Hiyama, Sho Yokoe, Tomomi Yamazaki, Teisuke Takita, Kiyoshi Yasukawa
{"title":"Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region.","authors":"Yutaro Narukawa,&nbsp;Mako Kandabashi,&nbsp;Tongyang Li,&nbsp;Misato Baba,&nbsp;Haruka Hara,&nbsp;Kenji Kojima,&nbsp;Kei Iida,&nbsp;Takayoshi Hiyama,&nbsp;Sho Yokoe,&nbsp;Tomomi Yamazaki,&nbsp;Teisuke Takita,&nbsp;Kiyoshi Yasukawa","doi":"10.1093/protein/gzab006","DOIUrl":"https://doi.org/10.1093/protein/gzab006","url":null,"abstract":"<p><p>Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) is widely used in research and clinical diagnosis. Improvement of MMLV RT thermostability has been an important topic of research for increasing the efficiency of cDNA synthesis. In this study, we attempted to increase MMLV RT thermostability by introducing a disulfide bridge in its RNase H region using site-directed mutagenesis. Five variants were designed, focusing on the distance between the two residues to be mutated into cysteine. The variants were expressed in Escherichia coli and purified. A551C/T662C was determined to be the most thermostable variant.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzab006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25566738","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
The evolution and engineering of enzyme activity through tuning conformational landscapes. 通过调整构象景观的酶活性的进化和工程。
IF 2.4 4区 生物学
Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab009
Adam M Damry, Colin J Jackson
{"title":"The evolution and engineering of enzyme activity through tuning conformational landscapes.","authors":"Adam M Damry,&nbsp;Colin J Jackson","doi":"10.1093/protein/gzab009","DOIUrl":"https://doi.org/10.1093/protein/gzab009","url":null,"abstract":"<p><p>Proteins are dynamic molecules whose structures consist of an ensemble of conformational states. Dynamics contribute to protein function and a link to protein evolution has begun to emerge. This increased appreciation for the evolutionary impact of conformational sampling has grown from our developing structural biology capabilities and the exploration of directed evolution approaches, which have allowed evolutionary trajectories to be mapped. Recent studies have provided empirical examples of how proteins can evolve via conformational landscape alterations. Moreover, minor conformational substates have been shown to be involved in the emergence of new enzyme functions as they can become enriched through evolution. The role of remote mutations in stabilizing new active site geometries has also granted insight into the molecular basis underpinning poorly understood epistatic effects that guide protein evolution. Finally, we discuss how the growth of our understanding of remote mutations is beginning to refine our approach to engineering enzymes.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38911037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
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