Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline.

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

Monte Carlo Thompson sampling-guided design for antibody engineering.

Antibodies are one of the predominant treatment modalities for various diseases. To improve the characteristics of a lead antibody, such as antigen-binding affinity and stability, we conducted comprehensive substitutions and exhaustively explored their sequence space. However, it is practically unfeasible to evaluate all possible combinations of mutations owing to combinatorial explosion when multiple amino acid residues are incorporated. It was recently reported that a machine-learning guided protein engineering approach such as Thompson sampling (TS) has been used to efficiently explore sequence space in the framework of Bayesian optimization. For TS, over-exploration occurs when the initial data are biasedly distributed in the vicinity of the lead antibody. We handle a large-scale virtual library that includes numerous mutations. When the number of experiments is limited, this over-exploration causes a serious issue. Thus, we conducted Monte Carlo Thompson sampling (MTS) to balance the exploration-exploitation trade-off by defining the posterior distribution via the Monte Carlo method and compared its performance with TS in antibody engineering. Our results demonstrated that MTS largely outperforms TS in discovering desirable candidates at an earlier round when over-exploration occurs on TS. Thus, the MTS method is a powerful technique for efficiently discovering antibodies with desired characteristics when the number of rounds is limited.

Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.

The construction of custom libraries is critical for rational protein engineering and directed evolution. Array-synthesized oligo pools of thousands of user-defined sequences (up to ∼350 bases in length) have emerged as a low-cost commercially available source of DNA. These pools cost ≤10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next-generation protein and pathway engineering strategies, such as sequence-function mapping, enzyme minimization, or de-novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community.

Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.

In-silico investigation towards the non-invasive optical detection of blood lactate.

This paper uses Monte Carlo simulations to investigate the interaction of short-wave infrared (SWIR) light with vascular tissue as a step toward the development of a non-invasive optical sensor for measuring blood lactate in humans. The primary focus of this work was to determine the optimal source-detector separation, penetration depth of light at SWIR wavelengths in tissue, and the optimal light power required for reliable detection of lactate. The investigation also focused on determining the non-linear variations in absorbance of lactate at a few select SWIR wavelengths. SWIR photons only penetrated 1.3 mm and did not travel beyond the hypodermal fat layer. The maximum output power was only 2.51% of the input power, demonstrating the need for a highly sensitive detection system. Simulations optimized a source-detector separation of 1 mm at 1684 nm for accurate measurement of lactate in blood.

Towards antigen-specific Tregs for type 1 diabetes: Construction and functional assessment of pancreatic endocrine marker, HPi2-based chimeric antigen receptor.

Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing ß cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse ß cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8+ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4+ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.

Hybrid MC/MD for protein design.

Computational protein design relies on simulations of a protein structure, where selected amino acids can mutate randomly, and mutations are selected to enhance a target property, such as stability. Often, the protein backbone is held fixed and its degrees of freedom are modeled implicitly to reduce the complexity of the conformational space. We present a hybrid method where short molecular dynamics (MD) segments are used to explore conformations and alternate with Monte Carlo (MC) moves that apply mutations to side chains. The backbone is fully flexible during MD. As a test, we computed side chain acid/base constants or pKa's in five proteins. This problem can be considered a special case of protein design, with protonation/deprotonation playing the role of mutations. The solvent was modeled as a dielectric continuum. Due to cost, in each protein we allowed just one side chain position to change its protonation state and the other position to change its type or mutate. The pKa's were computed with a standard method that scans a range of pH values and with a new method that uses adaptive landscape flattening (ALF) to sample all protonation states in a single simulation. The hybrid method gave notably better accuracy than standard, fixed-backbone MC. ALF decreased the computational cost a factor of 13.

Engineering Cell-Free Protein Synthesis for High-Yield Production and Human Serum Activity Assessment of Asparaginase: Toward On-Demand Treatment of Acute Lymphoblastic Leukemia.

Acute lymphocytic leukemia (ALL) is a common childhood cancer in the United States, with over 6000 new cases diagnosed each year. Administration of bacterial asparaginase (ASNase) has improved survival rates to nearly 80%, however these therapeutics have high incidence of immunological neutralization and serum activity must be monitored for most effective treatment regimens. Here, a 72% improvement in cell-free protein synthesis (CFPS) of FDA approved l-asparaginase (crisantaspase) is demonstrated by employing an aspartate-fed-batch reactor format. A CFPS-based ASNase activity assay as a tool for therapeutic regimentation and production quality control is also presented. This work suggests that shelf-stable and low-cost Escherichia coli-based CFPS reactions may be employed on-demand to 1) synthesize biologics on-site for patient administration, 2) verify biologic activity for dosage calculations, and 3) monitor therapeutic activity in human serum during the treatment regimen. The combination of both therapeutic production and activity assessment introduces a concept of synergistic utility for bacterial cell lysates in modern medical treatment. Indeed, recent work with CFPS biosensors supports a not-too-distant future when shelf-stable E. coli CFPS systems are used to diagnose, treat, and monitor treatment of diseases in the clinical setting.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

An efficient, step-economical strategy for the design of functional metalloproteins.

The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. Although numerous strategies have been used to create new metalloproteins, pre-existing knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (metal active sites by covalent tethering or MASCoT) in which folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naive protein-protein interfaces. Metalloproteins generated using this strategy uniformly bind a wide array of first-row transition metal ions (MnII, FeII, CoII, NiII, CuII, ZnII and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for MnII to 50 fM for CuII). MASCoT readily affords coordinatively unsaturated metal centres-including a penta-His-coordinated non-haem Fe site-and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands such as nitric oxide to the interfacial metal centre.

Industrial uses of phospholipases: current state and future applications.

Phospholipids play a central role in all living organisms. Phospholipases, the enzymes aimed at modifying phospholipids, are consequently widespread in nature and play diverse roles, from lipid metabolism and cellular signaling in eukaryotes to virulence and nutrient acquisition in microbes. Phospholipases catalyze the hydrolysis of one or more ester or phosphodiester bonds of glycerophospholipids. The use of phospholipases with industrial purposes has constantly increased over the last 30 years. This demand is rapidly growing given the ongoing improvements in protein engineering and the reduction of enzymes manufacturing costs, making them suitable for industrial use. Here, a general overview of phopholipases A, B, C, and D and their industrial application is presented along with potential new uses for these enzymes. We draw attention to commercial phospholipases used to improve the emulsifying properties of products in the baking, egg, and dairy industries. On the other hand, the improvement of oil degumming by phospholipases is thoroughly analyzed. Moreover, recent developments in enzymatic biodiesel production and the use of phospholipases for the synthesis of phospholipids with pharmaceutical or nutritional value are reviewed.

Metabolic Engineering of a Homoserine-Derived Non-Natural Pathway for the De Novo Production of 1,3-Propanediol from Glucose.

Engineering a homoserine-derived non-natural pathway allows heterologous production of 1,3-propanediol (1,3-PDO) from glucose without adding expensive vitamin B12. Due to the lack of efficient enzymes to catalyze the deamination of homoserine and the decarboxylation of 4-hydroxy-2-ketobutyrate, the previously engineered strain can only produce 51.5 mg/L 1,3-PDO using homoserine and glucose as cosubstrates. In this study, we systematically screened the enzymes from different protein families to catalyze the two corresponding reactions and further optimized the selected enzymes by protein engineering. Together with the improvement of homoserine supply by systematic metabolic engineering, an engineered Escherichia coli strain with an optimal combination of aspartate transaminase ( aspC) from E. coli, pyruvate decarboxylase ( pdc) from Zymomonas mobiliz, and alcohol dehydrogenase yqhD from E. coli can produce 0.32 g/L 1,3-PDO from glucose in shake flask cultivation. The titer of 1,3-PDO was further increased to 0.49 g/L or 0.63 g/L by introducing a point mutation of I472A into pdc gene or constructing a fusion protein between aspC and pdc. This study lays the basis for developing a potential process for 1,3-PDO production from sugars without using expensive coenzyme B12.

[Biomanufacturing of monoclonal antibodies]. / La bioproduction des anticorps monoclonaux.

Antibody-based drugs are an increasingly important part of the therapeutic arsenal against a wide variety of medical conditions. As the number of commercial products and pipeline candidates grows, a crucial issue facing the industry is the current and future state of biomanufacturing. The productivity of the protein expression platforms, along with the performance of the technologies impacting upstream and downstream bioprocessing, are critical factors affecting the cost and time of therapeutic antibody development and commercialization. Cell engineering strategies are being used to improve the production of antibodies and to better control their quality in terms of posttranslational modifications, in particular with regards to their glycosylation state, as this can influence their therapeutic activity. Additionally, the advance of "omics" technologies have recently given rise to new possibilities in improving these expression platforms. We review here the various advances in biomanufacturing essential to the continued growth of the therapeutic antibody market.

TITLE: La bioproduction des anticorps monoclonaux. ABSTRACT: Les anticorps monoclonaux font désormais partie intégrante de l'arsenal thérapeutique pour une multitude de maladies. Étant donné le nombre croissant de produits commerciaux et de candidats en développement, l'état actuel des systèmes de bioproduction est une préoccupation majeure de l'industrie. La productivité des plateformes d'expression, ainsi que la performance des technologies utilisées dans les procédés en amont et en aval, sont des facteurs critiques qui ont un impact sur le coût et la durée du développement des anticorps thérapeutiques. De multiples stratégies de génie cellulaire peuvent être utilisées pour l'amélioration de la production des anticorps et pour un contrôle accru des modifications post-traductionnelles, comme la glycosylation, particulièrement importante, car elle peut avoir un effet prononcé sur l'activité thérapeutique des anticorps. Les avancées des techniques « omiques ¼ rendent maintenant possibles de nouvelles approches pour l'amélioration de ces plateformes. Nous passons ici en revue les progrès en bioproduction essentiels à la croissance continue du marché des anticorps thérapeutiques.

Fast and Flexible Synthesis of Combinatorial Libraries for Directed Evolution.

Directed evolution (DE) is a powerful tool for optimizing an enzyme's properties toward a particular objective, such as broader substrate scope, greater thermostability, or increased kcat. A successful DE project requires the generation of genetic diversity and subsequent screening or selection to identify variants with improved fitness. In contrast to random methods (error-prone PCR or DNA shuffling), site-directed mutagenesis enables the rational design of variant libraries and provides control over the nature and frequency of the encoded mutations. Knowledge of protein structure, dynamics, enzyme mechanisms, and natural evolution demonstrates that multiple (combinatorial) mutations are required to discover the most improved variants. To this end, we describe an experimentally straightforward and low-cost method for the preparation of combinatorial variant libraries. Our approach employs a two-step PCR protocol, first producing mutagenic megaprimers, which can then be combined in a "mix-and-match" fashion to generate diverse sets of combinatorial variant libraries both quickly and accurately.

Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2.

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500 µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native amino acid types for residues that contact the antigen in the native structures was 72% in simulations performed in the presence of the antigen and 48% in simulations performed without the antigen, for an ARR of 1.5. For the non-contacting residues, the ARR was 1.08. This shows that the sequence profiles are able to maintain the amino acid types of these conserved, buried sites, while recovery of the exposed, contacting residues requires the presence of the antigen-antibody interface. We tested RAbD experimentally on both a lambda and kappa antibody-antigen complex, successfully improving their affinities 10 to 50 fold by replacing individual CDRs of the native antibody with new CDR lengths and clusters.

Facilitating circular permutation using Restriction Free (RF) cloning.

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.

Converting Enzymes into Tools of Industrial Importance.

BACKGROUND: Enzymes have applications in numerous biotechnological products and processes that are commonly used in the production of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes, however, are optimized to function under physiological conditions. Any change in reaction conditions results in their activity as well as stability being compromised. Hence, most of the natural biomolecules are not suitable for industrial applications. Modifications are required to develop efficient and successful reagents as per demand. Protein engineering can be applied to cope up with these situations. METHODS: This review describes some of the novel uses/unusual properties of enzymes as biological catalysts. It explains the different ways in which enzymes can be and have been used under non-native conditions. Different strategies have been discussed regarding stabilization of enzyme as well optimum conditions of its uses in different industries. The following patents databases were consulted: European Patent Office (EPO), the United States Patent and Trademark Office (USPTO), Patent scope Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: The review illustrates the width of the umbrella of applications covered by biocatalysts. Employing the tools of solvent and protein engineering, viz. non-aqueous media, additives, immobilization, mutagenesis, to name a few; biotechnology has been able to make enzyme catalyzed processes an essential components of the industrialist's armoury. CONCLUSION: The article lists a number of successful examples, both of patented technology as well as biocatalysts which are currently being used in the industry, to highlight the accomplishments of technologies which have been adopted till now for making enzyme technology industrially viable.

A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping.

There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.