Protein engineering | EPFL Graph Search

Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017. There are two general strategies for protein engineering: rational protein design and directed evolution. These methods are not mutually exclusive; researchers will often apply both. In the future, more detailed knowledge of protein structure and function, and advances in high-throughput screening, may greatly expand the abilities of protein engineering. Eventually, even unnatural amino acids may be included, via newer methods, such as expanded genetic code, that allow encodin

Developing structure-oriented models for enzymatic hydrolysis of lignocellulosic biomass

Jessica Charlène Rohrbach

With the unsustainable use of fossil fuels increasing strains on human institutions and ecosystems, the development of a renewable energy alternative is of paramount importance. Second generation biorefineries, based on the production of fuels and chemicals from lignocellulosic biomass, appear as attractive alternative to their non-renewable counterparts. Within the multiplicity of existing valorisation routes, enzymatic hydrolysis of lignocellulosic biomass into its constituent sugars has generated considerable interest, notably in the context of biofuels production. However, major hurdles stemming from the intricate structure of the lignocellulosic feedstocks still impede large-scale deployment of this process. The multiplicity of highly intertwined spatiotemporal factors impacting the enzymatic hydrolysis of lignocellulosic biomass represents a challenge to rationally design efficient hydrolysis process. Here, we propose a theory-based modelling framework relying on pore-diffusion and surface reaction to explore the effects of recognised bottlenecks on the enzymatic hydrolysis of lignocellulosic substrates. The model is based on a set of partial differential equations describing the evolution of the substrate morphology to investigate the interplay between experimental conditions and the physical characteristics of biomass particles as the reaction proceeds. The overall quantity of cellulase present in the hydrolysis mixture is carefully considered to investigate its interplay with the available accessible cellulose surface. Also, non-uniformity in terms of cellulose accessibility and cellulose digestibility are introduced in the model to weight their influence on observed hydrolysis rates. Finally, deactivation mechanisms are considered through unproductive adsorption of cellulases on both cellulose and lignin fractions, with the existence of such phenomena alleged to be critical in the efficiency of the hydrolysis process. Based on predictions of our model, we were able to confirm the critical role of cellulose accessibility, as defined by the combination of particle size, porosity and accessible cellulose surface, in dictating early reaction rates for a range of pretreated beech wood substrates. While high biomass loading should be favoured to improve enzyme penetration in the substrates, high enzyme loadings going beyond the initial number of cellulose adsorption appeared beneficial in notably two cases: (i) to promote internal diffusion in large particles and (ii) counteract undesired enzyme adsorption on lignin. For the latter, a relatively low increase in enzyme loading was sufficient to offset the resulting slowdown. We also showed that the existence of structural heterogeneities, and in particular non-uniform pore volume distribution within the lignocellulosic samples, contribute to the rate slowdown observed at later stage of the hydrolysis, while not explaining it in its entirety. Unproductive adsorption to cellulose, coupled to decrease in the cellulase efficiency at the cellulose surface, appeared as major contributor to the rate slowdown. Overall, we show how the use of a theory-based model can help decouple and evaluate the effects of key factors in the enzymatic hydrolysis of lignocellulosic biomass. As such, our model can help pave the way towards efficient integrated rational design strategies for enzyme process engineering for biomass conversion.

EPFL2021

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Matthew Christopher Blackburn

The research described within this thesis is primarily motivated by two fields of science with reversed, yet complementary, approaches to addressing the same essential objective: manipulating and understanding the complex program of life. Whereas synthetic biology has to do with the bottom-up construction of living systems from a library of âpartsâ, systems biology takes a top-down, reverse-engineering analysis of the same processes within functioning cells to elucidate the crucial elements and interactions. Both perspectives have led to important contributions in learning how biological systems operate. Discoveries from these studies can have far-reaching implications, notably in medicine, manufacturing, energy, and the environment. One exciting outcome of the research efforts within synthetic biology is the ability to design genetic constructs encoding proteins with novel, optimized functions. Although advances have been made in predicting how a proteinâs amino acid sequence relates to its higher order structural form and behavior, experimental methods for protein engineering remain invaluable for evaluating computationally derived designs. Unfortunately, the design-build-test cycle for rational protein development is commonly a time, labor and resource intensive endeavor. This is primarily due to limitations in the ability to generate libraries of gene variants and bottlenecks in cell-based expression and quantitative screening of protein products. This thesis describes the development of a solid-phase gene assembly technique and its integration with microfluidic protein analysis to accelerate the prototyping of novel protein designs. The gene assembly method utilizes commodity-scale, chemically manufactured DNA, and operates without the need for ligase or restriction enzymes. As a bench top process amenable to scale up, the modularity of the assembly process allows the efficient and economical generation of expression-ready gene variants. We directly coupled this gene assembly pipeline to a microfluidic device to enable high-throughput, on-chip, cell-free protein expression, purification, and characterization. By circumventing molecular cloning and cell-based steps, the lag time between protein design and quantitative analysis was dramatically reduced. As a proof of concept, this protein engineering platform was applied towards the construction of over 400 artificial, engineered variants of C2H2 zinc finger (ZF) proteins. ZF protein domains are the most prevalent form of transcription factor (TF) in humans, and are found throughout the tree of life. They provide a convenient structure for refactoring due to their relatively small size and composability, and are an ideal model for exploring the biophysics of TF-DNA specificity. The ability to engineer ZF proteins makes them useful as programmable, DNA-targeting units for applications in biotechnology and synthetic biology. We demonstrate that although ZFPs can be readily engineered to recognize a particular DNA target, engineering the precise binding energy landscape remains a challenge. Additionally, we show that ZF-DNA binding affinity can be tuned independently of sequence specificity. Together, these results demonstrate the versatility of the coupled gene assembly and microfluidic analysis platform as a new tool for rational protein engineering.

EPFL2016

Introducing tolerance to foreign and partly foreign proteins by erythrocyte binding and artificial glycosylations

Kym Lorenz Brünggel

Over the last few years, protein drugs have steadily gained importance in the clinic. Compared to small molecule drugs, protein drugs are taken up by antigen presenting cells, which degrade the proteins into peptides that are then presented on major histocompatability complex I and II to CD8+ respectively CD4+ T-cells. CD4+ T-cells are then able to activate B-cells, which upon activation will produce anti-drug antibodies. While anti-drug antibodies have mostly been viewed as a safety risk leading to drug injection related side effects, they have recently been proven to diminish the efficacy of the treatment. High antibody titers against Humira, a clinically approved monoclonal antibody used to treat rheumatoid arthritis, have been associated with low serum drug concentration and high disease activity score, ultimately leading to treatment failure. Two approaches to induce tolerance were developed in the Hubbell lab. One approach utilizes protein variants that are designed to bind to circulating erythrocytes. As 1 % of erythrocytes undergo apoptosis each day and are taken up by antigen presenting cells, along with the bound protein, they are presented to the immune system in a non-inflammatory tolerogenic fashion. Immunological tolerance is thereby induced against the apoptotic erythrocyte debris and the associated bound protein. The second approach uses synthetic glucose, galactose or mannose polymers conjugated to the target protein. The polymers target C-type lectins, which are known to clear apoptotic debris, leading to presentation of the protein by hepatic antigen presenting cells in a tolerogenic liver microenvironment. This thesis investigates the potential of these approaches to induce tolerance towards protein drugs. Arylsulfatase B was used as a model protein to investigate the potential of tolerance induction by targeting circulating erythrocytes, as 97% of patients receiving arylsulfatase B develop anti-drug antibodies. An erythrocyte binding variant of arylsufatase B was created by chemically conjugating the erythrocyte binding peptide ERY1 to arylsulfatse B. In mice receiving two doses of ERY1-arylsulfatase B one week apart followed by weekly doses of arylsulfatase B a significant delay of four week in the production of anti-drug Antibodies was found when compared to mice receiving weekly doses of only arylsulfatase B. To investigate the ability of synthetic glyco-polymers to induce tolerance towards protein drugs we used asparaginase as model protein. Between 37.5 % to 75% of patients treated with asparaginase develop anti-drug antibodies. Administering three intravenous injections of asparaginase-conjugated to glucose polymers before eight weeks of treatment with asparaginase significantly reduced anti-drug antibody titers by a factor of 125 when compared to animals only receiving eight weeks of treatment with asparaginase. Three pre-injections with asparaginase conjugated to the galactose or mannose polymer led to a reduction in anti-drug antibody titers by a factor of 12 respectively 400 after 5 weeks of treatment with asparaginase when compared to mice receiving only asparaginase for 5 weeks. These results indicates that synthetic glyco polymers, especially mannose and glucose based ones, are able to induce long term tolerance towards asparaginase. Synthetic glucose and mannose polymers therefore represent an excellent molecular approach to induce tolerance towards protein drugs.

EPFL2018