Phi29 DNA polymerase is widely used for DNA amplification through rolling-circle replication or multiple displacement amplification. Here, we performed completely in vitro artificial evolution of phi29 DNA polymerase by combining the in vitro compartmentalization and the gene expression-coupled rolling-circle replication of a circular DNA encoding the polymerase. We conducted the experiments in six

The linear extrapolation method to determine protein stability from denaturant-induced unfolding experiments is based on the observation that the free energy of unfolding is often a linear function of the denaturant concentration. The value in the absence of denaturant is then estimated by extrapolation from this linear relationship. Parameters and their confidence intervals are typically estimated

The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, 'ribosome pegging', to exclude common unwanted mutations that benefit host cells at the expense of heterologous

Computer-guided library generation is a plausible strategy to optimize antibodies. Herein, we report the improvement of the affinity of a single-domain camelid antibody for its antigen using such approach. We first conducted experimental and computational alanine scanning to describe the precise energetic profile of the antibody–antigen interaction surface. Based on this characterization, we hypothesized

Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the

Anionic surfactants denature proteins at low millimolar concentrations, yet little is known about the underlying molecular mechanisms. Here, we undertake 1-μs-long atomistic molecular dynamics simulations of the denaturation of acyl coenzyme A binding protein (ACBP) and compare our results with previously published and new experimental data. Since increasing surfactant chain length is known to lead

Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase

In the above article the symbols for ‘nanomolar’ and ‘micromolar’ were incorrectly expanded during typesetting to read ‘nanometer’ and ‘micrometer’ respectively. These have now been corrected to reflect their original intended meaning.

Intrinsically disordered proteins (IDPs) contribute to a multitude of functions. De novo design of IDPs should open the door to modulating functions and phenotypes controlled by these systems. Recent design efforts have focused on compositional biases and specific sequence patterns as the design features. Analysis of the impact of these designs on sequence-function relationships indicates that individual

The adaptive immune system of cartilaginous fish (Elasmobranchii), comprising of classical hetero-tetrameric antibodies, is enhanced through the presence of a naturally occurring homodimeric antibody-like immunoglobulin-the new antigen receptor (IgNAR). The binding site of the IgNAR variable single-domain (VNAR) offers advantages of reduced size (<1/10th of classical immunoglobulin) and extended binding

The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity

Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase

To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica, the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved

The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered

FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B)

Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants

Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having

Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered

The tachykinin 2 receptor (NK2R) plays critical roles in gastrointestinal, respiratory and mental disorders and is a well-recognized target for therapeutic intervention. To date, therapeutics targeting NK2R have failed to meet regulatory agency approval due in large part to the limited characterization of the receptor-ligand interaction and downstream signaling. Herein, we report a protein engineering

The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral

The Dynameomics project contains native state and unfolding simulations of 807 protein domains, where each domain is representative of a different metafold; these metafolds encompass ~97% of protein fold space. There is a long-standing question in structural biology as to whether proteins in the same fold family share the same folding/unfolding characteristics. Using molecular dynamics simulations

Aggregation of islet amyloid polypeptide (IAPP) into islet amyloid results in β-cell toxicity in human type 2 diabetes. To determine the effect of islet amyloid formation on gene expression, we performed ribonucleic acid (RNA) sequencing (RNA-seq) analysis using cultured islets from either wild-type mice (mIAPP), which are not amyloid prone, or mice that express human IAPP (hIAPP), which develop amyloid

The polypeptide amylin is responsible for islet amyloid in type 2 diabetes, a process which contributes to β-cell death in the disease. The role of the N-terminal region of amylin in amyloid formation is relatively unexplored, although removal of the disulfide bridged loop between Cys-2 and Cys-7 accelerates amyloid formation. We examine the des Lys-1 variant of human amylin (h-amylin), a variant which

Islet amyloid is a pathologic feature of type 2 diabetes (T2D) that is associated with β-cell loss and dysfunction. These amyloid deposits form via aggregation of the β-cell secretory product islet amyloid polypeptide (IAPP) and contain other molecules including the heparan sulfate proteoglycan perlecan. Perlecan has been shown to bind amyloidogenic human IAPP (hIAPP) via its heparan sulfate glycosaminoglycan

The pyruvate dehydrogenase complex (PDHc) from Escherichia coli is a large protein complex consisting of multiple copies of the pyruvate dehydrogenase (E1ec), dihydrolipoamide acetyltransferase (E2ec) and dihydrolipoamide dehydrogenase (E3ec). The N-terminal domain (NTD, residues 1-55) of E1ec plays a critical role in the interaction between E1ec and E2ec and the whole PDHc activity. Using circular

Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target

Prostate apoptosis response-4 (Par-4) is a tumor suppressor which protects against neoplastic transformation. Remarkably, Par-4 is capable of inducing apoptosis selectively in cancer cells without affecting the normal cells. In this study, we found that recombinant Par-4 protein had limited serum persistence in mice that may diminish its anti-tumor activity in vivo. To improve the in vivo performance

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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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