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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...
September 20, 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)...
September 19, 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
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...
July 10, 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...
July 10, 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...
July 5, 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
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
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
Jennifer I Lai, Deeptak Verma, Chris Bailey-Kellogg, Margaret E Ackerman
Structure-based approaches to antigen design utilize insights from antibody (Ab):antigen interactions and a refined understanding of protective Ab responses to engineer novel antigens presenting epitopes with conformations relevant to eliciting or discovering protective humoral responses. For human immunodeficiency virus-1 (HIV-1), one model of protection is provided by broadly neutralizing Abs (bnAbs) against epitopes present in the closed prefusion trimeric conformation of HIV-1 envelope glycoprotein, such as the variable loops 1-2 (V1V2) apex...
April 1, 2018: Protein Engineering, Design & Selection: PEDS
Mitesh Nagar, Himank Kumar, Stephen L Bearne
Mandelate racemase (MR) serves as a paradigm for our understanding of enzyme-catalyzed deprotonation of a carbon acid substrate. To facilitate structure-function studies on MR using non-natural amino acid substitutions, we engineered the Cys92Ser/Cys264Ser variant (dmMR) as a platform for introducing Cys residues at specific locations for subsequent covalent modification. While the highly reactive thiol of Cys furnishes a site for chemical modification, site-specificity requires that other Cys residues be non-reactive or replaced by a non-reactive amino acid, especially if chemical modification is conducted under denaturing conditions...
April 1, 2018: Protein Engineering, Design & Selection: PEDS
Maike Lenz, Silvia Fademrecht, Mahima Sharma, Jürgen Pleiss, Gideon Grogan, Bettina M Nestl
We report the exploration of the evolutionary relationship between imine reductases (IREDs) and other dehydrogenases. This approach is informed by the sequence similarity between these enzyme families and the recently described promiscuous activity of IREDs for the highly reactive carbonyl compound 2,2,2-trifluoroacetophenone. Using the structure of the R-selective IRED from Streptosporangium roseum (R-IRED-Sr) as a model, β-hydroxyacid dehydrogenases (βHADs) were identified as the dehydrogenases most similar to IREDs...
April 1, 2018: Protein Engineering, Design & Selection: PEDS
Harun F Ozbakir, Kristen E Garcia, Scott Banta
Enzymatic biocatalysis can be limited by the necessity of soluble cofactors. Here, we introduced PEGylated nicotinamide adenine dinucleotide (NAD(H)) swing arms to two covalently fused dehydrogenase enzymes to eliminate their nicotinamide cofactor requirements. A formate dehydrogenase and cytosolic malate dehydrogenase were connected via SpyCatcher-SpyTag fusions. Bifunctionalized polyethylene glycol chains tethered NAD(H) to the fusion protein. This produced a formate:malate oxidoreductase that exhibited cofactor-independent ping-pong kinetics with predictable Michaelis constants...
April 1, 2018: Protein Engineering, Design & Selection: PEDS
S Palikša, G Alzbutas, R Skirgaila
Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display technique for in vitro evolution of the Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT) that would be able to perform efficient full-length cDNA synthesis in PCR buffer optimized for Thermus aquaticus DNA polymerase...
March 1, 2018: Protein Engineering, Design & Selection: PEDS
Annalee W Nguyen, Kevin C Le, Jennifer A Maynard
Discovery of monoclonal antibodies is most commonly performed using phage or yeast display but mammalian cells are used for production because of the complex antibody structure, including the multiple disulfide bonds and glycosylation, required for function. As this transition between host organisms is often accompanied by impaired binding, folding or expression, development pipelines include laborious plate-based screening or engineering strategies to adapt an antibody to mammalian expression. To circumvent these problems, we developed a plasmid-based Fab screening platform on Chinese hamster ovary (CHO) cells which allows for antibody selection in the production host and in the presence of the same post-translational modifications as the manufactured product...
March 1, 2018: Protein Engineering, Design & Selection: PEDS
Nicolás Palopoli, Nicolás S González Foutel, Toby J Gibson, Lucía B Chemes
Pocket proteins retinoblastoma (pRb), p107 and p130 are negative regulators of cellular proliferation and multifunctional proteins regulating development, differentiation and chromatin structure. The retinoblastoma protein is a potent tumor suppressor mutated in a wide range of human cancers, and oncogenic viruses often interfere with cell cycle regulation by inactivating pRb. The LxCxE and pRb AB groove short linear motifs (SLiMs) are key to many pocket protein mediated interactions including host and viral partners...
March 1, 2018: Protein Engineering, Design & Selection: PEDS
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