Rational evolution for altering the ligand preference of estrogen receptor alpha.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

Site-Selective Functionalization of Nanobodies Using Intein-Mediated Protein Ligation for Innovative Bioconjugation.

An expression strategy is presented in order to produce nanobodies modified with a clickable alkyne functionality at their C-terminus via the intein-mediated protein ligation (IPL) technique. The protocol focuses on the cytoplasmic expression and extraction of a nanobody-intein-chitin binding domain (CBD) fusion protein in E. coli SHuffle® T7 cells, in the commonly used Luria-Bertani (LB) medium. The combination of these factors results in a high yield and nearly complete alkynation of the nanobody at its C-terminus via IPL. The resulting alkynated nanobodies retain excellent binding capacity toward the nanobody targeted antigen. The presented protocol benefits from time- and cost-effectiveness and allows for a feasible upscaling of functionalized (here alkynated) nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to (1) novel biosurface applications that demand for homogeneously oriented nanobodies having their active site fully accessible for target (e.g., biomarker) binding, and (2) innovative applications such as localized drug delivery and image guided surgery by covalent "click" chemistry coupling of these alkynated nanobodies to a multitude of azide-containing counterparts as there are drug containing polymers and contrast labeling agents.

Enzyme-mediated hydrogel encapsulation of single cells for high-throughput screening and directed evolution of oxidoreductases.

Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors. Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol cross-coupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within ~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to ~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H 2 O 2 -producing enzymes many folds.

Steered molecular dynamics simulations reveal the role of Ca2+ in regulating mechanostability of cellulose-binding proteins.

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.

Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement.

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4'-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects.

Magnetic Separation of Elastin-like Polypeptide Receptors for Enrichment of Cellular and Molecular Targets.

Protein-conjugated magnetic nanoparticles (mNPs) are promising tools for a variety of biomedical applications, from immunoassays and biosensors to theranostics and drug-delivery. In such applications, conjugation of affinity proteins (e.g., antibodies) to the nanoparticle surface many times compromises biological activity and specificity, leading to increased reagent consumption and decreased assay performance. To address this problem, we engineered a biomolecular magnetic separation system that eliminates the need to chemically modify nanoparticles with the capture biomolecules or synthetic polymers of any kind. The system consists of (i) thermoresponsive magnetic iron oxide nanoparticles displaying poly(N-isopropylacrylamide) (pNIPAm), and (ii) an elastin-like polypeptide (ELP) fused with the affinity protein Cohesin (Coh). Proper design of pNIPAm-mNPs and ELP-Coh allowed for efficient cross-aggregation of the two distinct nanoparticle types under collapsing stimuli, which enabled magnetic separation of ELP-Coh aggregates bound to target Dockerin (Doc) molecules. Selective resolubilization of the ELP-Coh/Doc complexes was achieved under intermediate conditions under which only the pNIPAm-mNPs remained aggregated. We show that ELP-Coh is capable of magnetically separating and purifying nanomolar quantities of Doc as well as eukaryotic whole cells displaying the complementary Doc domain from diluted human plasma. This modular system provides magnetic enrichment and purification of captured molecular targets and eliminates the requirement of biofunctionalization of magnetic nanoparticles to achieve bioseparations. Our streamlined and simplified approach is amenable for point-of-use applications and brings the advantages of ELP-fusion proteins to the realm of magnetic particle separation systems.

Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies.

Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets-a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.

An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications.

Protein engineering for directed immobilization.

Much effort has been put into the optimization of the functional activity of proteins. For biosensors this protein functional optimization will increase the biosensor's sensitivity and/or selectivity. However, the strategy chosen for the immobilization of the proteins to the sensor surface might be equally important for the development of sensor surfaces that are optimally biologically active. Several studies published in recent years show that the oriented immobilization of the bioactive molecules improves the sensor's properties. In this review, we discuss the state of the art of the different protein immobilization strategies that are commonly used today with a special focus on biosensor applications. These strategies include nonspecific immobilization techniques either by physical adsorption, by covalent coupling, or by specific immobilization via site-specifically introduced tags or bio-orthogonal chemistry. The different tags and bio-orthogonal chemistry available and the techniques to site-specifically introduce these groups in proteins are also discussed.