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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
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...
May 3, 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
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 5, 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
Boudhayan Bandyopadhyay, Yoav Peleg
Circular permutation is a powerful tool to test the role of topology in protein folding and function. Previous methods for generating circular permutants were based on rearranging gene elements using restriction enzymes-based cloning. Here, we present a Restriction Free (RF) approach to achieve circular permutation which is faster and more cost-effective.
March 1, 2018: Protein Engineering, Design & Selection: PEDS
Zhuo Yang, Mingjuan Du, Wei Wang, Xiu Xin, Peixiang Ma, Hongkai Zhang, Richard A Lerner
It has been observed that converting scFv formatted antibodies to full-length IgG often associates with loss of affinity. We aim to address this issue in this paper by establishing an integrated affinity maturation method applying yeast display technology platform. To demonstrate that, we employed a human thrombopoietin receptor targeting antibody named 3D9 which was identified previously from a combinational antibody library in scFv-Fc fusion protein form. We have observed that significant potency loss happened when 3D9 was transformed to full-length IgG form...
February 21, 2018: Protein Engineering, Design & Selection: PEDS
Andrew K D Younger, Peter Y Su, Andrea J Shepard, Shreya V Udani, Thaddeus R Cybulski, Keith E J Tyo, Joshua N Leonard
Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors...
February 1, 2018: Protein Engineering, Design & Selection: PEDS
D Sussman, L Westendorf, D W Meyer, C I Leiske, M Anderson, N M Okeley, S C Alley, R Lyon, R J Sanderson, P J Carter, D R Benjamin
Antibody-drug conjugates (ADCs) are fulfilling the promise of targeted therapy with meaningful clinical success. An intense research effort is directed towards improving pharmacokinetic profiles, toxicity and chemical stability of ADCs. The majority of ADCs use amide and thioether chemistry to link potent cytotoxic agents to antibodies via endogenous lysine and cysteine residues. While maleimide-cysteine conjugation is used for many clinical stage ADC programs, maleimides have been shown to exhibit some degree of post-conjugation instability...
February 1, 2018: Protein Engineering, Design & Selection: PEDS
Malgorzata Figiel, Piotr Bonarek, Andrzej Górecki, Sebastian D Pawlak, Bartlomiej Zerek, Beata Checinska, Jerzy Pieczykolan, Marta Dziedzicka-Wasylewska
The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences...
February 1, 2018: Protein Engineering, Design & Selection: PEDS
Maho Yagi-Utsumi, Arunima Sikdar, Toshiya Kozai, Rintaro Inoue, Masaaki Sugiyama, Takayuki Uchihashi, Hirokazu Yagi, Tadashi Satoh, Koichi Kato
Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments...
January 1, 2018: Protein Engineering, Design & Selection: PEDS
Lei Li, Tian Xie, Zhongchuan Liu, Hong Feng, Ganggang Wang
CotA protein from Bacillus subtilis is of laccase activity. The solubility of recombinant CotA is low, which hinders its application. In this study, histidine tag position optimization and hydrophilic engineering were applied to increase the yield and activity of CotA protein. The results showed that the protein yield of CotA with his tag at C-terminal (CH6-CotA) was four times of that of NH6-CotA (His tag at N-terminal). Then, 23 single mutants were constructed by substitutions of hydrophobic residues with hydrophilic amino acids...
January 1, 2018: Protein Engineering, Design & Selection: PEDS
Yu Zhou, Hao Zou, Christina Yau, Lequn Zhao, Steven C Hall, Daryl C Drummond, Shauna Farr-Jones, John W Park, Christopher C Benz, James D Marks
We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies...
January 1, 2018: Protein Engineering, Design & Selection: PEDS
Ewa Oclon, Gili Solomon, Zvi Hayouka, Tomer Meir Salame, Vincent Goffin, Arieh Gertler
To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology...
January 1, 2018: Protein Engineering, Design & Selection: PEDS
Oliviero Carugo
Amino acids and their properties are variably distributed in proteins and different compositions determine all protein features, ranging from solubility to stability and functionality. Gini index, a tool to estimate distribution uniformity, is widely used in macroeconomics and has numerous statistical applications. Here, Gini index is used to analyze the distribution of hydrophobicity in proteins and to compare hydrophobicity distribution in globular and intrinsically disordered proteins. Based on the analysis of carefully selected high-quality data sets of proteins extracted from the Protein Data Bank (http://www...
December 1, 2017: Protein Engineering, Design & Selection: PEDS
M Hinrichsen, M Lenz, J M Edwards, O K Miller, S G J Mochrie, P S Swain, U Schwarz-Linek, L Regan
We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP...
December 1, 2017: Protein Engineering, Design & Selection: PEDS
Nancy L Goicochea, Maria Garnovskaya, Mary G Blanton, Grace Chan, Richard Weisbart, Michael B Lilly
Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody...
December 1, 2017: Protein Engineering, Design & Selection: PEDS
Camille Villequey, Xu-Dong Kong, Christian Heinis
Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type...
November 1, 2017: Protein Engineering, Design & Selection: PEDS
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