Protein Engineering, Design and Selection最新文献_第2页

Engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis 通过合理设计和诱变工程表达抗白细胞介素-13抗体

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzx001

B. Popovic, Suzanne J Gibson, Tarik Senussi, Sara Carmen, S. Kidd, T. Slidel, I. Strickland, Jianqing Xu, J. Spooner, A. Lewis, N. Hudson, Lorna Mackenzie, J. Keen, B. Kemp, C. Hardman, D. Hatton, T. Wilkinson, T. Vaughan, D. Lowe

{"title":"Engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis","authors":"B. Popovic, Suzanne J Gibson, Tarik Senussi, Sara Carmen, S. Kidd, T. Slidel, I. Strickland, Jianqing Xu, J. Spooner, A. Lewis, N. Hudson, Lorna Mackenzie, J. Keen, B. Kemp, C. Hardman, D. Hatton, T. Wilkinson, T. Vaughan, D. Lowe","doi":"10.1093/protein/gzx001","DOIUrl":"https://doi.org/10.1093/protein/gzx001","url":null,"abstract":"High levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. Here, we describe two related antibodies, Ab001 and Ab008, where Ab001 shows a markedly lower level of expression relative to Ab008 when stably expressed in Chinese hamster ovary cells. We use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the VL CDR2 of Ab001. We adopted two approaches to improve the expression of Ab001. First, we used mutagenesis to change single amino-acid residues in the Ab001 VL back to the equivalent Ab008 residues but this resulted in limited improvements in expression. In contrast when we used an in silico structure-based design approach to generate a set of five individual single-point variants in a discrete region of the VL, all exhibited significantly improved expression relative to Ab001. The most successful of these, D53N, exhibited a 25-fold increase in stable transfectants relative to Ab001. The functional potency of these VL-modified antibodies was unaffected. We expect that this in silico engineering strategy can be used to improve the expression of other antibodies and proteins.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"26 1","pages":"303–311"},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82274398","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein 通过与嗜热宿主蛋白的组合插入融合稳定环状芽孢杆菌木聚糖酶

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzw081

Vandan Shah, B. Pierre, Tamari Kirtadze, Seung-Yeol Shin, J. Kim

{"title":"Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein","authors":"Vandan Shah, B. Pierre, Tamari Kirtadze, Seung-Yeol Shin, J. Kim","doi":"10.1093/protein/gzw081","DOIUrl":"https://doi.org/10.1093/protein/gzw081","url":null,"abstract":"High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX). Specifically, we used an engineered transposon to construct a combinatorial library of randomly inserted BCX into PfMBP. The library was then subjected to functional screening to identify successful PfMBP-BCX insertion complexes, PfMBP-BCX161 and PfMBP-BCX165, displaying substantially improved kinetic stability at elevated temperatures compared to unfused BCX and other controls. Results from subsequent characterizations were consistent with the view that lowered aggregation of BCX and reduced conformational flexibility at the termini was responsible for increased thermal stability. Our stabilizing approach neither sacrificed xylanase activity nor required changes in the BCX amino acid sequence. Overall, the current study demonstrated the benefit of combinatorial insertional fusion to PfMBP as a systematic tool for the creation of enzymatically active and thermostable BCX variants.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"309 1","pages":"281–290"},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82539105","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display 结合动物免疫和酵母展示制备人双特异性普通轻链抗体

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzw077

Simon Krah, C. Schröter, Carla Eller, Laura Rhiel, Nicolas Rasche, J. Beck, Carolin Sellmann, Ralf Günther, L. Toleikis, B. Hock, H. Kolmar, Stefan Becker

{"title":"Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display","authors":"Simon Krah, C. Schröter, Carla Eller, Laura Rhiel, Nicolas Rasche, J. Beck, Carolin Sellmann, Ralf Günther, L. Toleikis, B. Hock, H. Kolmar, Stefan Becker","doi":"10.1093/protein/gzw077","DOIUrl":"https://doi.org/10.1093/protein/gzw077","url":null,"abstract":"Bispecific antibodies (bsAbs) pave the way for novel therapeutic modes of action along with potential benefits in several clinical applications. However, their generation remains challenging due to the necessity of correct pairings of two different heavy and light chains and related manufacturability issues. We describe a generic approach for the generation of fully human IgG-like bsAbs. For this, heavy chain repertoires from immunized transgenic rats were combined with either a randomly chosen common light chain or a light chain of an existing therapeutic antibody and screened for binders against tumor-related targets CEACAM5 and CEACAM6 by yeast surface display. bsAbs with subnanomolar affinities were identified, wherein each separate binding arm mediated specific binding to the respective antigen. Altogether, the described strategy represents a combination of in vivo immunization with an in vitro selection method, which allows for the integration of existing therapeutic antibodies into a bispecific format.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"1 1","pages":"291–301"},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76727885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 37

Ligand-induced conformational changes in prolyl oligopeptidase: a kinetic approach 配体诱导的脯氨酰寡肽酶的构象变化:动力学方法

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzw079

R. V. Elzen, E. Schoenmakers, Inger Brandt, P. Veken, A. Lambeir

引用次数: 8

Secretion of functional formate dehydrogenase in Pichia pastoris 毕赤酵母中功能性甲酸脱氢酶的分泌

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzx010

M. Takacs, O. Makhlynets, Patricia L. Tolbert, I. Korendovych

引用次数: 5

Modification of the peroxygenative: peroxidative activity ratio in the unspecific peroxygenase from Agrocybe aegerita by structure-guided evolution 结构导向进化对绿草草非特异性过氧酶过氧活性比的修饰

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzw073

D. Maté, M. A. Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, M. Alcalde

{"title":"Modification of the peroxygenative: peroxidative activity ratio in the unspecific peroxygenase from Agrocybe aegerita by structure-guided evolution","authors":"D. Maté, M. A. Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, M. Alcalde","doi":"10.1093/protein/gzw073","DOIUrl":"https://doi.org/10.1093/protein/gzw073","url":null,"abstract":"Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values. The full ensemble of segments (43% of UPO sequence) was subjected to focused evolution by the Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) method in Saccharomyces cerevisiae. Five independent mutant libraries were screened in terms of P:p ratio and thermostability. We identified several variants that harbored substitutions at positions 120 and 320 with a strong enhancement in the P:p ratio albeit at the cost of stability. The most thermostable mutant of this process (S226G with an increased T50 of 2°C) was subjected to further combinatorial saturation mutagenesis on Thr120 and Thr320 yielding a collection of variants with modified P:p ratio and recovered stability. Our results seem to indicate the coexistence of several oxidation sites for peroxidative and peroxygenative activities in UPO.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"27 1","pages":"189–196"},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81774134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 24

Probing the influence of non-covalent contact networks identified by charge density analysis on the oxidoreductase BacC 探讨电荷密度分析鉴定的非共价接触网络对氧化还原酶BacC的影响

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzx006

K. Perinbam, H. Balaram, T. N. Guru Row, B. Gopal

{"title":"Probing the influence of non-covalent contact networks identified by charge density analysis on the oxidoreductase BacC","authors":"K. Perinbam, H. Balaram, T. N. Guru Row, B. Gopal","doi":"10.1093/protein/gzx006","DOIUrl":"https://doi.org/10.1093/protein/gzx006","url":null,"abstract":"Bacillus subtilis BacC is an oxidoreductase involved in the biosynthesis of the potent antibiotic bacilysin. The crystal structure of BacC was determined at 1.19 Å resolution. An experimental charge density approach was used to calculate non-covalent interactions within the monomer and across the dimeric interface of BacC. This interaction network, in turn, enabled an analysis of non-covalently connected paths that span the protein structure. One of the pathways of non-covalent interactions was examined by mutational analysis. Biochemical analysis of BacC mutants with potential disruptions in non-covalent interactions along this path revealed that residues that form nodes in pathways of non-covalent interactions influence catalytic activity more than others in a similar chemical environment. Furthermore, we note that nodes in the non-covalent interaction networks are co-localized with compensatory mutation sites identified by multiple sequence alignment of proteins with low sequence similarity to BacC. Put together, this analysis supports the hypothesis that non-covalent nodes represent conserved structural features that can impact the catalytic activity of an enzyme.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"46 1","pages":"263–270"},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78795870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1 墨西哥利什曼原虫scp2 -硫酶(2型)的晶体学底物结合研究:氧阴离子孔-1的独特特征

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzw080

R. Harijan, T. Kiema, Shahan M Syed, Imran Qadir, M. Mazet, F. Bringaud, P. Michels, R. Wierenga

{"title":"Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1","authors":"R. Harijan, T. Kiema, Shahan M Syed, Imran Qadir, M. Mazet, F. Bringaud, P. Michels, R. Wierenga","doi":"10.1093/protein/gzw080","DOIUrl":"https://doi.org/10.1093/protein/gzw080","url":null,"abstract":"C\u0000Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"60 1","pages":"225–233"},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84007099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Collective repacking reveals that the structures of protein cores are uniquely specified by steric repulsive interactions 集体重新包装揭示了蛋白质核心的结构是由空间排斥性相互作用唯一指定的

Protein Engineering, Design and Selection Pub Date : 2017-02-15 DOI: 10.1093/protein/gzx011

Jennifer C. Gaines, Alejandro Virrueta, D. A. Buch, S. Fleishman, C. O’Hern, Lynne Regan

{"title":"Collective repacking reveals that the structures of protein cores are uniquely specified by steric repulsive interactions","authors":"Jennifer C. Gaines, Alejandro Virrueta, D. A. Buch, S. Fleishman, C. O’Hern, Lynne Regan","doi":"10.1093/protein/gzx011","DOIUrl":"https://doi.org/10.1093/protein/gzx011","url":null,"abstract":"Abstract Protein core repacking is a standard test of protein modeling software. A recent study of six different modeling software packages showed that they are more successful at predicting side chain conformations of core compared to surface residues. All the modeling software tested have multicomponent energy functions, typically including contributions from solvation, electrostatics, hydrogen bonding and Lennard–Jones interactions in addition to statistical terms based on observed protein structures. We investigated to what extent a simplified energy function that includes only stereochemical constraints and repulsive hard-sphere interactions can correctly repack protein cores. For single residue and collective repacking, the hard-sphere model accurately recapitulates the observed side chain conformations for Ile, Leu, Phe, Thr, Trp, Tyr and Val. This result shows that there are no alternative, sterically allowed side chain conformations of core residues. Analysis of the same set of protein cores using the Rosetta software suite revealed that the hard-sphere model and Rosetta perform equally well on Ile, Leu, Phe, Thr and Val; the hard-sphere model performs better on Trp and Tyr and Rosetta performs better on Ser. We conclude that the high prediction accuracy in protein cores obtained by protein modeling software and our simplified hard-sphere approach reflects the high density of protein cores and dominance of steric repulsion.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"11 1","pages":"387 - 394"},"PeriodicalIF":0.0,"publicationDate":"2017-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73163427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Engineering potent long-acting variants of the Wnt inhibitor DKK2 设计有效的长效Wnt抑制剂DKK2变体

Protein Engineering, Design and Selection Pub Date : 2017-02-09 DOI: 10.1093/protein/gzx007

Richelle Sopko, Joshua W. Mugford, A. Lehmann, R. Shapiro, M. Rushe, Abhishek Kulkarni, Joe Worrall, Joseph Amatucci, Dingyi Wen, N. Pederson, Brenda K. Minesinger, J. Arndt, B. Pepinsky

{"title":"Engineering potent long-acting variants of the Wnt inhibitor DKK2","authors":"Richelle Sopko, Joshua W. Mugford, A. Lehmann, R. Shapiro, M. Rushe, Abhishek Kulkarni, Joe Worrall, Joseph Amatucci, Dingyi Wen, N. Pederson, Brenda K. Minesinger, J. Arndt, B. Pepinsky","doi":"10.1093/protein/gzx007","DOIUrl":"https://doi.org/10.1093/protein/gzx007","url":null,"abstract":"Abstract Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"42 1","pages":"359 - 372"},"PeriodicalIF":0.0,"publicationDate":"2017-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76004797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2