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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...
January 29, 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...
January 23, 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...
January 23, 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...
January 18, 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.
December 29, 2017: 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...
December 28, 2017: 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...
December 28, 2017: 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...
December 28, 2017: 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...
December 21, 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 7, 2017: 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 5, 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 29, 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
Lei Tao, Dandan Li, Yonghong Li, Xinchang Shi, Junzhi Wang, Chunming Rao, Yingqi Zhang
As human uricase has been silenced during evolution, counterparts from other species become an alternative for the treatment of hyperuricemia. Candida uricase is a promising option among them, but its aggregation propensity remains a major obstacle to clinical use. In this study, we designed two mutations according to homology-modeled 3D structure of Candida uricase: Cys249Ser substitution and C-terminal Leu deletion. The wild-type uricase and three mutants containing either or both of the mutations were expressed in Escherichiacoli BL21 and validated by mass spectrometry...
November 17, 2017: Protein Engineering, Design & Selection: PEDS
Yu Gao, Jian-Jun Li, Lanyan Zheng, Yuguang Du
Versatile peroxidase (VP) from Pleurotus eryngii is a high redox potential peroxidase. It has aroused great biotechnological interest due to its ability to oxidize a wide range of substrates, but its application is still limited due to low pH and thermal stability. Since CiP (Coprinopsis cinerea peroxidase) and PNP (peanut peroxidase) exhibited higher pH and thermal stability than VP, several motifs, which might contribute to their pH and thermal stability, were identified through structure and sequence alignment...
November 7, 2017: Protein Engineering, Design & Selection: PEDS
B M Xavier, E Hildebrandt, F Jiang, H Ding, J C Kappes, I L Urbatsch
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a plasma membrane chloride channel protein that regulates vertebrate fluid homeostasis. The inefficiency of wild type human CFTR protein folding/trafficking is exacerbated by genetic mutations that can cause protein misfolding in the endoplasmic reticulum (ER) and subsequent degradation. This project investigates small changes in protein sequence that can alter the thermal stability of the large multi-domain CFTR protein. We target a conserved 70-residue α-subdomain located in the first nucleotide-binding domain that hosts the common misfolding mutation ∆F508...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
M Amitay, M Goldstein
DEEPSAM is a relatively new global optimization algorithm aimed to predict the structure of bio-molecules from sequence, without any additional preliminary assumption. It is an evolutionary algorithm whose mutation operators are built by hybridizing the diffusion equation method, molecular dynamics simulated annealing, and a quasi-Newton local minimization method. The goal of this study was to evaluate the structure prediction capabilities of DEEPSAM by running it upon NMR structures of linear peptides (10-20 residues)...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
J Wong, X Chen, K Truong
Since tobacco etch virus protease (TEVp) has a high specificity and efficiency in cleaving its target substrates, many groups have attempted to engineer conditional control of its activity. Temperature induction is widely used for modulating gene function because it has fast temporal response, good penetrability and applicability to many model organisms. Here, we engineered a temperature sensitive TEVp (tsTEVp) by using N-terminal truncations to TEVp that achieved efficient proteolysis on a timescale of 4 h after 30°C induction, while remaining relatively inactive at 37°C...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
Casey K Hua, Albert T Gacerez, Charles L Sentman, Margaret E Ackerman
As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
Paul W H I Parren, James S Huston
No abstract text is available yet for this article.
September 1, 2017: Protein Engineering, Design & Selection: PEDS
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