Polymer-based Protein Engineering Research Articles

Site-specific modification of N-terminal α-amino groups of succinylated collagen

Polymer based protein engineering provides an attractive strategy to endow novel properties to protein and overcome the inherent limitations of both counterparts. The exquisite control of site and density of attached polymers on the proteins is crucial for the bioactivities and properties of the protein-polymer bioconjugates, but is still a challenge. Collagen is the major structural protein in extracellular matrix of animals. Based on the advancements of polymer-based protein engineering, collagen bioconjugates has been widely fabricated and applied as biomaterials. However, the site-specific synthesis of well-defined collagen-polymer bioconjugates is still not achieved. Herein, a versatile strategy for the specific modification of N-terminal α-amino groups in collagen was developed. Firstly, all reactive amino groups of tropocollagen (collagen with telopeptides) were protected by succinic anhydride. Then, the telopeptides were digested to give the active N-terminal α-amino groups, which were subsequently attached with poly(N-isopropylacrylamide) (PNIPAAm) via “grafting from” method based on the atom transfer radical polymerization (ATRP). The site-specific N-terminal PNIPAAm modified succinylated collagen was prepared and its structure, thermal responsive behaviour, and properties was explored.

A comprehensive analysis in one run - in-depth conformation studies of protein-polymer chimeras by asymmetrical flow field-flow fractionation.

Polymer-based protein engineering has enabled the synthesis of a variety of protein–polymer conjugates that are widely applicable in therapeutic, diagnostic and biotechnological industries. Accurate characterizations of physical–chemical properties, in particular, molar masses, sizes, composition and their dispersities are critical parameters that determine the functionality and conformation of protein–polymer conjugates and are important for creating reproducible manufacturing processes. Most of the current characterization techniques suffer from fundamental limitations and do not provide an accurate understanding of a sample's true nature. In this paper, we demonstrate the advantage of asymmetrical flow field-flow fractionation (AF4) coupled with multiple detectors for the characterization of a library of complex, zwitterionic and neutral protein–polymer conjugates. This method allows for determination of intrinsic physical properties of protein–polymer chimeras from a single, rapid measurement.

Tertiary Structure-Based Prediction of How ATRP Initiators React with Proteins.

The growth of polymers from the surface of proteins via controlled radical polymerization depends on the attachment of small molecule initiators to amino acid residues. Our ability to control and harness the power of polymer-based protein engineering is reliant on the accuracy of prediction where and how fast atom transfer radical polymerization (ATRP) initiators will react with a protein surface. We performed a systematic characterization of the reaction between a bromine-functionalized N-hydroxysuccinimide amine-reactive ATRP initiator and the amino groups in lysozyme and chymotrypsin. The tertiary structures of the proteins were used to predict computationally α-amino group and lysine side-chain accessibility and analyze the chemical and structural environment of the amino groups. To predict reactivity from accessibility calculations, a probe radius that resembled the size of the initiator molecule was used. Experimental data showed that the rate of initiator-protein modification at each amine site was related to surface accessibility but not the pKa of amino groups. Further refinements of the prediction of where the initiator modified the protein and in what sequence were achieved by considering the local environment of each amino group.

Intramolecular Electron Transfer through Poly-Ferrocenyl Glucose Oxidase Conjugates to Carbon Electrodes: 1. Sensor Sensitivity, Selectivity and Longevity

Enzyme-based amperometric biosensors measure target analyte concentrations through the transduction of enzymatically generated electrons into a detectable current at an electrode surface. To achieve efficient electron transfer, many such systems rely on the use of diffusive electron mediators or intricate, nanostructured electrode materials, which can limit applicability. Herein, we report on the development and characterization of enzyme-based glucose biosensors operating using a combination of intramolecular electron transfer and electron self-exchange through ferrocene-containing redox mediators covalently coupled directly to glucose oxidase (GOX). To accomplish this, we applied polymer-based protein engineering to grow poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide (pFcAc) via atom-transfer radical polymerization from initiator molecules attached to the GOX surface. The use of these GOX-pFcAc conjugates allowed the fabrication of simple, paper-based glucose biosensors through crosslinking with the carrier protein human serum albumin (HSA) within a viscous chitosan solution and drop casting onto carbon paper strips. Chit-GOX-pFcAc-HSA-carbon paper biosensors yielded a glucose sensitivity of 0.33±0.01μAmM−1cm−2 and exhibited selectivity when challenged with other sugars. The ease of sensor fabrication further afforded a tailorable response through variation of GOX-pFcAc concentration upon crosslinking and drop casting. The reported approach shows the glucose sensing capabilities of a system that merges the characteristics of bioelectronics fabricated using redox-containing polymer networks with those based on the surface modification of enzymes with electron relays.

Intramolecular Electron Transfer through Poly-Ferrocenyl Glucose Oxidase Conjugates to Carbon Electrodes: 2. Mechanistic Understanding of Long-Term Stability

Enzyme-based biosensors have garnered intense research interest due to the catalytic properties and physiologic applicability of enzymes. However, the use of these biological catalysts also introduced persistent lifetime limiting stability issues that hinder device longevity. We report on the identification and understanding of sources of long-term stability loss within a glucose oxidase (GOX)-based biosensor. Through polymer-based protein engineering (PBPE), we grew poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide (pFcAc) directly from initiator sites at the GOX surface via atom-transfer radical polymerization. The resulting GOX-pFcAc conjugates were crosslinked along with human serum albumin (HSA) within chitosan (Chit)-containing solution and drop cast onto carbon paper strips. Chit-GOX-pFcAc-HSA-carbon paper biosensors generated current by intramolecular electron transfer and electron self-exchange through the GOX-pFcAc conjugates as well as through oxygen mediated electron transfer under varied cell potentials and solution conditions. A systematic characterization method identified the leading sources of instability within the enzyme-polymer conjugate system. We found that a physically crosslinked network prevented GOX leaching from the functionalized electrode. Pre-wetting the electrode also increased current outputs. A reduction in ferrocene-mediated current generation resulted from instability of the ferrocenium ion produced during operation. This issue could be solved through the use of freely diffusing ferrocene, which indicated a path toward improvement for enzyme-polymer conjugate systems with carefully engineered redox characteristics.

ATRP-grown protein-polymer conjugates containing phenylpiperazine selectively enhance transepithelial protein transport

Despite its patient-friendliness, the oral route is not yet a viable strategy for the delivery of biomacromolecular therapeutics. This is, in part, due to the large size of proteins, which greatly limits their absorption across the intestinal epithelium. Although chemical permeation enhancers can improve macromolecular transport, their positive impact is often accompanied by toxicity. One element potentially contributing to this toxicity is the lack of co-localization of the enhancer with the protein drug, which can result in non-specific permeation of the intestine as well as enhancer overdosing in some areas due to non-uniform distribution. To circumvent these issues, this study describes a new way of increasing protein permeability via a polymer conjugation process that co-localizes permeation enhancer with the protein. Based on previous reports demonstrating the utility of 1-phenylpiperazine as an intestinal permeation enhancer, we synthesized protein-polymer conjugates with a phenylpiperazine-containing polymer using polymer-based protein engineering. A novel phenylpiperazine acrylamide monomer was synthesized and chain extended using atom transfer radical polymerization from the model protein bovine serum albumin (BSA). At non-cytotoxic doses, the protein-polymer conjugates induced a dose dependent reduction in the trans-epithelial electrical resistance of Caco-2 monolayers and an impressive ~30-fold increase in BSA permeability. Furthermore, this permeability increase was selective, as the permeability of the small molecule calcein co-incubated with the protein-polymer conjugate increased only 5-fold. Together, these data represent an important first step in the development of protein polymer conjugates that facilitate selective protein transport across membranes that are typically impermeable to macromolecules.

Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates.

The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.

Polymer-Based Protein Engineering: Synthesis and Characterization of Armored, High Graft Density Polymer-Protein Conjugates.

Atom transfer radical polymerization (ATRP) from the surface of a protein can generate remarkably dense polymer shells that serve as armor and rationally tune protein function. Using straightforward chemistry, it is possible to covalently couple or display multiple small molecule initiators onto a protein surface. The chemistry is fine-tuned to be sequence specific (if one desires a single targeted site) at controlled density. Once the initiator is anchored on the protein surface, ATRP is used to grow polymers on protein surface, in situ. The technique is so powerful that a single-protein polymer conjugate molecule can contain more than 90% polymer coating by weight. If desired, stimuli-responsive polymers can be "grown" from the initiated sites to prepare enzyme conjugates that respond to external triggers such as temperature or pH, while still maintaining enzyme activity and stability. Herein, we focus mainly on the synthesis of chymotrypsin-polymer conjugates. Control of the number of covalently coupled initiator sites by changing the stoichiometric ratio between enzyme and the initiator during the synthesis of protein-initiator complexes allowed fine-tuning of the grafting density. For example, very high grafting density chymotrypsin conjugates were prepared from protein-initiator complexes to grow the temperature-responsive polymers, poly(N-isopropylacrylamide), and poly[N,N'-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate]. Controlled growth of polymers from protein surfaces enables one to predictably manipulate enzyme kinetics and stability without the need for molecular biology-dependent mutagenesis.

Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell

Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a “grafting-from” approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective “wiring” of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm−2) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization.

Rational tailoring of substrate and inhibitor affinity via ATRP polymer-based protein engineering.

Atom transfer radical polymerization (ATRP)-based protein engineering of chymotrypsin with a cationic polymer was used to tune the substrate specificity and inhibitor binding. Poly(quaternary ammonium) was grown from the surface of the enzyme using ATRP after covalent attachment of a protein reactive, water-soluble ATRP-initiator. This "grafting from" conjugation approach generated a high density of cationic ammonium ions around the biocatalytic core. Modification increased the surface area of the protein over 40-fold, and the density of modification on the protein surface was approximately one chain per 4 nm(2). After modification, bioactivity was increased at low pH relative to the activity of the native enzyme. In addition, the affinity of the enzyme for a peptide substrate was increased over a wide pH range. The massively cationic chymotrypsin, which included up to 2000 additional positive charges per molecule of enzyme, was also more stable at extremes of temperature and pH. Most interestingly, we were able to rationally control the binding of two oppositely charged polypeptide protease inhibitors, aprotinin and the Bowman-Birk trypsin-chymotrypsin inhibitor from Glycine max, to the cationic derivative of chymotrypsin. This study expands upon our efforts to use polymer-based protein engineering to predictably engineer enzyme properties without the need for molecular biology.

Dramatically Increased pH and Temperature Stability of Chymotrypsin Using Dual Block Polymer-Based Protein Engineering

In this study, we report on multimodal temperature-responsive chymotrypsin-poly(sulfobetaine methacrylamide)-block-poly(N-isopropylacrylamide) (CT-pSBAm-block-pNIPAm) protein-polymer conjugates. Using polymer-based protein engineering (PBPE) with aqueous atom transfer radical polymerization (ATRP), we synthesized three different molecular weight CT-pSBAm-block-pNIPAm bioconjugates that responded structurally to both low and high temperature. In the block copolymer grown from the surface of the enzyme, upper critical solution temperature (UCST) phase transition was dependent on the chain length of the polymers in the conjugates, whereas lower critical solution temperature (LCST) phase transition was independent of molecular weight. Each CT-pSBAm-block-pNIPAm conjugate showed temperature dependent changes in substrate affinity and productivity when assayed from 0 to 40 °C. In addition, these conjugates showed higher stability to harsh conditions, including temperature, low pH, and protease degradation. Indeed, the PBPE-modified enzyme was active for over 8 h in the presence of a stomach protease at pH 1.0. Using PBPE, we created a dual zone shell surrounding each molecule of enzyme. The thickness of each zone of the shell was engineered to be separately responsive to temperature.

Tailoring enzyme activity and stability using polymer-based protein engineering

Polymer-based protein engineering (PBPE) offers an attractive method to predictably modify and enhance enzyme structure and function. Using polymers that respond to stimuli such as temperature and pH, enzyme activity and stability can be predictably modified without a dependence on molecular biology. Herein, we demonstrate that temperature responsive enzyme-polymer conjugates show increased stability while retaining bioactivity and substrate affinity. The bioconjugates were synthesized using a “grafting from” approach, where polymers were grown from a novel water-soluble initiator on the surface of a protein using atom transfer radical polymerization. Prior to polymer synthesis, the polymerization initiating molecule was covalently attached to surface accessible primary amines (lysine, N-terminal) of chymotrypsin, forming a macroinitiator. Poly(N-isopropylacrylamide) and poly[N,N’-dimethyl(methacryloylethyl) ammonium propane sulfonate] were grown separately from the initiator modified chymotrypsin. Both polymers were selected because of their temperature-dependent conformations. We observed that the enzyme-polymer conjugates retained temperature-dependent changes in conformation while still maintaining enzyme function. The conjugates exhibited dramatic increases in enzyme stability over a wide range of temperatures. We can now predictably manipulate enzyme kinetics and stability using polymer-based protein engineering without the need for molecular biology dependent mutagenesis.

Polymer-Based Protein Engineering Can Rationally Tune Enzyme Activity, pH-Dependence, and Stability

The attachment of inert polymers, such as polyethylene glycol, to proteins has driven the emergence of a multibillion dollar biotechnology industry. In all cases, proteins have been stabilized or altered by covalently coupling the pre-existing polymer to the surface of the protein. This approach is inherently limited by a lack of exquisite control of polymer architecture, site and density of attachment. Using a novel water-soluble atom transfer radical polymerization initiator, we have grown temperature- and pH-responsive polymers from the surface of a model protein, the enzyme chymotrypsin. Poly(2-(dimethylamino)ethyl methacrylate) changes in conformation with altered temperature and pH. Growing the polymer from the surface of chymotrypsin we were able to demonstrate that changes in temperature or pH can change predictably the conformation of the polymer surrounding the enzyme, which in turn enabled the rational tailoring of enzyme activity and stability. Using what we now term "Polymer-Based Protein Engineering", we have increased the activity and stability of chymotrypsin by an order of magnitude at pHs where the enzyme is usually inactive or unstable.