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Protein Engineering, Design & Selection: PEDS

Tian Jiang, P Douglas Renfrew, Kevin Drew, Noah Youngs, Glenn L Butterfoss, Richard Bonneau, Den Nis Shasha
A wide variety of protein and peptidomimetic design tasks require matching functional 3D motifs to potential oligomeric scaffolds. For example, during enzyme design, one aims to graft active-site patterns-typically consisting of 3-15 residues-onto new protein surfaces. Identifying protein scaffolds suitable for such active-site engraftment requires costly searches for protein folds that provide the correct side chain positioning to host the desired active site. Other examples of biodesign tasks that require similar fast exact geometric searches of potential side chain positioning include mimicking binding hotspots, design of metal binding clusters and the design of modular hydrogen binding networks for specificity...
November 8, 2018: Protein Engineering, Design & Selection: PEDS
Chang-Hao Wu, Cynthia A Ponir, Dominik K Haja, Michael W W Adams
The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100°C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H2/glucose) from sugars compared to cellular fermentations (4 H2/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme...
October 24, 2018: Protein Engineering, Design & Selection: PEDS
Ugur Pala, Berin Yelmazer, Meltem Çorbacioglu, Jouni Ruupunen, Jarkko Valjakka, Ossi Turunen, Baris Binay
Conversion of hydrogen carbonate to formate by mutants of Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In CtFDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduction) as a function of substrate concentration...
October 15, 2018: Protein Engineering, Design & Selection: PEDS
James S Huston
No abstract text is available yet for this article.
October 15, 2018: Protein Engineering, Design & Selection: PEDS
Michael J Rudolph, David J Vance, Simon Kelow, Siva Krishna Angalakurthi, Sophie Nguyen, Simon A Davis, Yinghui Rong, C Russell Middaugh, David D Weis, Roland Dunbrack, John Karanicolas, Nicholas J Mantis
Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2...
September 28, 2018: Protein Engineering, Design & Selection: PEDS
Sandeep Kumar, Kirk Roffi, Dheeraj S Tomar, David Cirelli, Nicholas Luksha, Danielle Meyer, Jeffrey Mitchell, Martin J Allen, Li Li
Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of MAB1 containing single point (V44K, E59S, E59T and E59Y) and double (V44KE59S, V44KE59T and V44KE59Y) mutations in its light chain were produced in Chinese Hamster Ovary (CHO) cells and purified by using platform processes employed during commercial scale production of monoclonal antibodies...
September 5, 2018: Protein Engineering, Design & Selection: PEDS
S-Y Lau, J W Siau, R M Sobota, C-I Wang, P Zhong, D P Lane, F J Ghadessy
Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold...
August 30, 2018: Protein Engineering, Design & Selection: PEDS
Joerg Thomas Regula, Sabine Imhof-Jung, Michael Mølhøj, Joerg Benz, Andreas Ehler, Alexander Bujotzek, Wolfgang Schaefer, Christian Klein
Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers...
August 28, 2018: Protein Engineering, Design & Selection: PEDS
Laura S Mitchell, Lucy J Colwell
Nanobodies (Nbs) are a class of antigen-binding protein derived from camelid immune systems, which achieve equivalent binding affinities and specificities to classical antibodies (Abs) despite being comprised of only a single variable domain. Here, we use a data set of 156 unique Nb:antigen complex structures to characterize Nb-antigen binding and draw comparison to a set of 156 unique Ab:antigen structures. We analyse residue composition and interactions at the antigen interface, together with structural features of the paratopes of both data sets...
July 25, 2018: Protein Engineering, Design & Selection: PEDS
Sudeep Karki, Prodeep Paudel, Celeste Sele, Alexander V Shkumatov, Tommi Kajander
Synaptic adhesion molecules play a crucial role in the regulation of synapse development and maintenance. Recently, several families of leucine-rich repeat (LRR) domain-containing neuronal adhesion molecules have been characterised, including netrin-G ligands, LRRTMs and the synaptic adhesion-like molecule (SALM) family proteins. Most of these are expressed at the excitatory glutamatergic synapses, and dysfunctions of these genes are genetically linked with cognitive disorders, such as autism spectrum disorders and schizophrenia...
June 12, 2018: Protein Engineering, Design & Selection: PEDS
Dana N Ashoor, Noureddine Ben Khalaf, Sonia Bourguiba-Hachemi, Maryam H Marzouq, M Dahmani Fathallah
The interaction between antibodies and Immune cells surface FcγRIIIa (CD16a) receptor triggers a variety of immune responses including antibody-dependent cell-mediated cytotoxicity, antibody neutralization, phagocytosis, inflammation and tissue injury. Recent studies showed that IgG1 upper hinge region and FcγRs polymorphism play a major role in the interaction with Fcγ receptors and in the stability of the immune complex hence, in mounting strong inflammatory response. To further investigate this issue, we developed a tool box of IgG1 Fc isoforms to depict the affinity between mutated IgG1 Fc regions and extracellular domain variants (V158F) of CD16a...
June 1, 2018: Protein Engineering, Design & Selection: PEDS
Rauda A Mohamed, Abu Bakar Salleh, Thean Chor Leow, Normi M Yahaya, Mohd Basyaruddin Abdul Rahman
A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region...
June 1, 2018: Protein Engineering, Design & Selection: PEDS
C S Frei, S Qian, P C Cirino
Customized transcription factors that control gene expression in response to small molecules can act as endogenous molecular biosensors and are valuable tools for synthetic biology. We previously engineered the Escherichia coli regulatory protein AraC to respond to non-native inducers such as D-arabinose and triacetic acid lactone. Those prior studies involved the construction and screening of individual 4- or 5-site saturation mutagenesis libraries, followed by iterative rounds of positive- and negative fluorescence-activated cell sorting (FACS)...
June 1, 2018: Protein Engineering, Design & Selection: PEDS
Matthew Carter Childers, Clare-Louise Towse, Valerie Daggett
Computational resources have contributed to the design and engineering of novel proteins by integrating genomic, structural and dynamic aspects of proteins. Non-canonical amino acids, such as d-amino acids, expand the available sequence space for designing and engineering proteins; however, the rotamer libraries for d-amino acids are usually constructed as the mirror images of l-amino acid rotamer libraries, an assumption that has not been tested. To this end, we have performed molecular dynamics (MD) simulations of model host-guest peptide systems containing d-amino acids...
June 1, 2018: Protein Engineering, Design & Selection: PEDS
Raiji Kawade, Hiroki Akiba, Kevin Entzminger, Toshiaki Maruyama, C J Okumura, Kouhei Tsumoto
Rabbit antibodies show unique structural characteristics in that kappa chains have an inter-domain disulfide bond between the variable and constant domains. Here we characterized this disulfide bond from physicochemical viewpoints both in stability and affinity. It was revealed that the disulfide bond contributed to the thermal stability of the antibody, but the affinity and mechanism of antigen recognition was not altered by the mutation. The present result expands the understanding of how rabbit antibodies with kappa light chains gain affinity under characteristic mechanism to gain thermal stability, and would give suggestions for the methods to artificially stabilize antibody molecules...
May 30, 2018: Protein Engineering, Design & Selection: PEDS
L Schwaigerlehner, M Pechlaner, P Mayrhofer, C Oostenbrink, R Kunert
Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments...
May 11, 2018: Protein Engineering, Design & Selection: PEDS
Daša Lipovšek, Irvith Carvajal, Alban J Allentoff, Anthony Barros, John Brailsford, Qiang Cong, Pete Cotter, Sanjeev Gangwar, Cris Hollander, Virginie Lafont, Wai Leung Lau, Wenying Li, Miguel Moreta, Steven O'Neil, Jason Pinckney, Michael J Smith, Julie Su, Christina Terragni, Michael A Wallace, Lifei Wang, Martin Wright, H Nicholas Marsh, James W Bryson
Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody-drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens-but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin-drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtration eliminates Adnectins from the bloodstream within minutes to hours, ensuring low exposure to normal tissues...
May 1, 2018: Protein Engineering, Design & Selection: PEDS
Marco Mravic, Hailin Hu, Zhenwei Lu, Joel S Bennett, Charles R Sanders, A Wayne Orr, William F DeGrado
Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbβ3 and αvβ3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5β1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite...
May 1, 2018: Protein Engineering, Design & Selection: PEDS
Roberto De Luca, Paul Kachel, Klara Kropivsek, Berend Snijder, Markus G Manz, Dario Neri
A novel dual-cytokine-antibody fusion protein, consisting of an antibody directed against CD38 [a tumor-associated antigen mainly expressed on the surface of multiple myeloma (MM) cells], simultaneously fused to both tumor necrosis factor ligand superfamily member 10 (TRAIL) and interleukin-2 (IL2), was designed, expressed and purified to homogeneity. The novel fusion protein, termed IL2-αCD38-αCD38-scTRAIL, was able to selectively recognize its cognate antigen expressed on the surface of MM and lymphoma cell lines, as evidenced by flow cytometry analysis...
May 1, 2018: Protein Engineering, Design & Selection: PEDS
Xiufeng Wu, Richard Yuan, Michael Bacica, Stephen J Demarest
The clinical success of monoclonal antibodies to treat diseases across nearly every therapeutic area has spurred advances in bispecific antibody technology with the goal of cost-effectively combining various therapies or providing novel mechanisms for disease intervention. Many novel bispecific antibodies are now in clinical development or the late pre-clinical setting. A new horizon exists for novel molecular entities with the ability to bind three or more antigens. Here we describe the production and characterization of novel trispecific antibody-like proteins denoted 'OrthoTsAbs' that self-assemble through the application of engineered antibody domain interfaces...
April 28, 2018: Protein Engineering, Design & Selection: PEDS
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