Protein Engineering Design & Selection最新文献_第9页

Toward the design of efficient transglycosidases: the case of the GH1 of Thermus thermophilus. 高效转糖苷酶的设计:以嗜热热菌GH1为例。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz032

B. David, P. Arnaud, C. Tellier, Y. Sanejouand

引用次数: 7

Dissecting the statistical properties of the linear extrapolation method of determining protein stability. 剖析了测定蛋白质稳定性的线性外推法的统计特性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa010

Kresten Lindorff-Larsen

引用次数: 5

Attempts to develop an enzyme converting DHIV to KIV. 试图开发一种将hiv转化为KIV的酶。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz042

Kenji Oki, Frederick S Lee, Stephen L Mayo

{"title":"Attempts to develop an enzyme converting DHIV to KIV.","authors":"Kenji Oki, Frederick S Lee, Stephen L Mayo","doi":"10.1093/protein/gzz042","DOIUrl":"https://doi.org/10.1093/protein/gzz042","url":null,"abstract":"Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 6","pages":"261-270"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz042","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37486616","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

Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion. 重组质粒的统计噪声可以通过核糖体蛋白基因缺失的补充来减弱。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa007

Ichiro Matsumura, Donian Chyong

{"title":"Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion.","authors":"Ichiro Matsumura, Donian Chyong","doi":"10.1093/protein/gzaa007","DOIUrl":"https://doi.org/10.1093/protein/gzaa007","url":null,"abstract":"The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, 'ribosome pegging', to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 10","pages":"433-441"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37867398","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: 0

Another week, another breakthrough: immunotherapy. 又一个星期，又一个突破:免疫疗法。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz045

James S Huston

引用次数: 0

Antibody humanization-the Influence of the antibody framework on the CDR-H3 loop ensemble in solution. 抗体人源化——抗体框架对溶液中CDR-H3环系的影响。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa004

Monica L Fernández-Quintero, Martin C Heiss, Klaus R Liedl

{"title":"Antibody humanization-the Influence of the antibody framework on the CDR-H3 loop ensemble in solution.","authors":"Monica L Fernández-Quintero, Martin C Heiss, Klaus R Liedl","doi":"10.1093/protein/gzaa004","DOIUrl":"10.1093/protein/gzaa004","url":null,"abstract":"Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the complementarity determining region (CDR)-H3 loop dynamics. We also studied a Fv without structural information of the anti-idiotypic antibody Ab2/3H6, because it completely lost its binding affinity upon superhumanization, as an example of a failed humanization. Enhanced sampling techniques in combination with molecular dynamics simulations allow to access micro- to milli-second timescales of the CDR-H3 loop dynamics and reveal kinetic and thermodynamic changes involved in the process of humanization. In most cases, we observe a reduced conformational diversity of the CDR-H3 loop when grafted on a human framework and find a conformational shift of the dominant CDR-H3 loop conformation in solution. A shallow side minimum of the conformational CDR-H3 loop ensemble attached to the murine framework becomes the dominant conformation in solution influenced by the human framework. Additionally, we observe in the case of the failed humanization that the potentially binding competent murine CDR-H3 loop ensemble in solution shows nearly no kinetical or structural overlap with the superhumanized variant, thus explaining the loss of binding.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 9","pages":"411-422"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098879/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37703280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Computer-guided library generation applied to the optimization of single-domain antibodies. 计算机引导文库生成应用于单结构域抗体的优化。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa006

Hiroki Akiba, Hiroko Tamura, Jose M M Caaveiro, Kouhei Tsumoto

引用次数: 6

Protein A superantigen: structure, engineering and molecular basis of antibody recognition. 蛋白A超抗原:结构、工程和抗体识别的分子基础。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz026

Ohan Mazigi, Peter Schofield, D. Langley, D. Christ

引用次数: 10

Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents. 针对人类治疗性Fc支架的高选择性IgNAR可变单结构域的分离及其作为定制生物处理试剂的应用。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa002

Magdalena J Buschhaus, Stefan Becker, Andrew J Porter, Caroline J Barelle

{"title":"Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents.","authors":"Magdalena J Buschhaus, Stefan Becker, Andrew J Porter, Caroline J Barelle","doi":"10.1093/protein/gzaa002","DOIUrl":"https://doi.org/10.1093/protein/gzaa002","url":null,"abstract":"The adaptive immune system of cartilaginous fish (Elasmobranchii), comprising of classical hetero-tetrameric antibodies, is enhanced through the presence of a naturally occurring homodimeric antibody-like immunoglobulin-the new antigen receptor (IgNAR). The binding site of the IgNAR variable single-domain (VNAR) offers advantages of reduced size (<1/10th of classical immunoglobulin) and extended binding topographies, making it an ideal candidate for accessing cryptic epitopes otherwise intractable to conventional antibodies. These attributes, coupled with high physicochemical stability and amenability to phage display, facilitate the selection of VNAR binders to challenging targets. Here, we explored the unique attributes of these single domains for potential application as bioprocessing reagents in the development of the SEED-Fc platform, designed to generate therapeutic bispecific antibodies. A panel of unique VNARs specific to the SEED homodimeric (monospecific) 'by-products' were isolated from a shark semi-synthetic VNAR library via phage display. The lead VNAR candidate exhibited low nanomolar affinity and superior selectivity to SEED homodimer, with functionality being retained upon exposure to extreme physicochemical conditions that mimic their applicability as purification agents. Ultimately, this work exemplifies the robustness of the semi-synthetic VNAR platform, the predisposition of the VNAR paratope to recognise novel epitopes and the potential for routine generation of tailor-made VNAR-based bioprocessing reagents.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 9","pages":"385-399"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37695454","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

Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR. 利用分子伴侣TAPBPR在哺乳动物细胞中产生可溶性pmhc - 1分子。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa015

Sara M O'Rourke, Giora I Morozov, Jacob T Roberts, Adam W Barb, Nikolaos G Sgourakis

{"title":"Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.","authors":"Sara M O'Rourke, Giora I Morozov, Jacob T Roberts, Adam W Barb, Nikolaos G Sgourakis","doi":"10.1093/protein/gzaa015","DOIUrl":"10.1093/protein/gzaa015","url":null,"abstract":"Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 12","pages":"525-532"},"PeriodicalIF":2.6,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451022/pdf/gzaa015.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38213003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0