Kristi L. Kiick

Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cells native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.

Polysaccharide-modified synthetic polymeric biomaterials.

This review presents an overview of polysaccharide‐conjugated synthetic polymers and their use in tissue‐engineered scaffolds and drug‐delivery applications. This topic will be divided into four categories: (1) polymeric materials modified with non‐mammalian polysaccharides such as alginate, chitin, and dextran; (2) polymers modified with mammalian polysaccharides such as hyaluronan, chondroitin sulfate, and heparin; (3) multi‐polysaccharide‐derivatized polymer conjugate systems; and (4) polymers containing polysaccharide‐mimetic molecules. Each section will discuss relevant conjugation techniques, analysis, and the impact of these materials as micelles, particles, or hydrogels used in in‐vitro and in‐vivo biomaterial applications.

Tunable Degradation of Maleimide–Thiol Adducts in Reducing Environments

Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here, we show that the resulting succinimide thioether formed by the Michael-type addition of thiols to N-ethylmaleimide (NEM), generally accepted as stable, undergoes retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature, offering a novel strategy for controlled release. Model studies ((1)H NMR, HPLC) of NEM conjugated to 4-mercaptophenylacetic acid (MPA), N-acetylcysteine, or 3-mercaptopropionic acid (MP) incubated with glutathione showed half-lives of conversion from 20 to 80 h, with extents of conversion from 20% to 90% for MPA and N-acetylcysteine conjugates. After ring-opening, the resultant succinimide thioether did not show retro and exchange reactions. The kinetics of the retro reactions and extent of exchange can be modulated by the Michael donors reactivity; therefore, the degradation of maleimide-thiol adducts could be tuned for controlled release of drugs or degradation of materials at time scales different than those currently possible via disulfide-mediated release. Such approaches may find a new niche for controlled release in reducing environments relevant in chemotherapy and subcellular trafficking.

Hybrid Multicomponent Hydrogels for Tissue Engineering

Artificial ECMs that not only closely mimic the hybrid nature of the natural ECM but also provide tunable material properties and enhanced biological functions are attractive candidates for tissue engineering applications. This review summarizes recent advances in developing multicomponent hybrid hydrogels by integrating modular and heterogeneous building blocks into well-defined, multifunctional hydrogel composites. The individual building blocks can be chemically, morphologically, and functionally diverse, and the hybridization can occur at molecular level or microscopic scale. The modular nature of the designs, combined with the potential synergistic effects of the hybrid systems, has resulted in novel hydrogel matrices with robust structure and defined functions.

Protein- and peptide-modified synthetic polymeric biomaterials

This review presents an overview on bio‐hybrid approaches of integrating the structural and functional features of proteins and peptides with synthetic polymers and the resulting unique properties in such hybrids, with a focus on bioresponsive/bioactive systems with biomaterials applications. The review is divided in two broad sections. First, we describe several examples of bio‐hybrids produced by combining versatile synthetic polymers with proteins/enzymes and drugs that have resulted in (1) hybrid materials based on responsive polymers, (2) responsive hydrogels based on enzyme‐catalyzed reactions, protein–protein interactions and protein–drug sensing, and (3) dynamic hydrogels based on conformational changes of a protein. Next, we present hybrids produced by combining synthetic polymers with peptides, classified based on the properties of the peptide domain: (1) peptides with different conformations, such as α‐helical, coiled‐coil, and β‐sheet; (2) peptides derived from structural protein domains such as silk, elastin, titin, and collagen; and (3) peptides with other biofunctional properties such as cell‐binding domains and enzyme‐recognized degradation domains.

Polysaccharide−Poly(ethylene glycol) Star Copolymer as a Scaffold for the Production of Bioactive Hydrogels

The production of polysaccharide-derivatized surfaces, polymers, and biomaterials has been shown to be a useful strategy for mediating the biological properties of materials, owing to the importance of polysaccharides for the sequestration and protection of bioactive proteins in vivo. We have therefore sought to combine the benefits of polysaccharide derivatization of polymers with unique opportunities to use these polymers for the production of bioactive, noncovalently assembled hydrogels. Accordingly, we report the synthesis of a heparin-modified poly(ethylene glycol) (PEG) star copolymer that can be used in the assembly of bioactive hydrogel networks via multiple strategies and that is also competent for the delivery of bioactive growth factors. A heparin-decorated polymer, synthesized by the reaction of thiol end-terminated four-arm star PEG (M(n) = 10 000) with maleimide functionalized low molecular weight heparin (LMWH, M(r) = 3000), has been characterized via (1)H NMR spectroscopy and size-exclusion chromatography; results indicate attachment of the LMWH with at least 73% efficiency. Both covalently and noncovalently assembled hydrogels can be produced from the PEG-LMWH conjugate. Viscoelastic noncovalently assembled hydrogels have been formed on the basis of the interaction of the PEG-LMWH with a PEG polymer bearing multiple heparin-binding peptide motifs. The binding and release of therapeutically important proteins from the assembled hydrogels have also been demonstrated via immunochemical assays, which demonstrate the slow release of basic fibroblast growth factor (bFGF) as a function of matrix erosion. The combination of these results suggests the opportunities for producing polymer-polysaccharide conjugates that can assemble into novel hydrogel networks on the basis of peptide-saccharide interactions and for employing these materials in delivery applications.

Heparin-functionalized polymeric biomaterials in tissue engineering and drug delivery applications.

Heparin plays an important role in many biological processes via its interaction with various proteins, and hydrogels and nanoparticles comprising heparin exhibit attractive properties, such as anticoagulant activity, growth factor binding, and antiangiogenic and apoptotic effects, making them great candidates for emerging applications. Accordingly, this review summarizes recent efforts in the preparation of heparin-based hydrogels and formation of nanoparticles, as well as the characterization of their properties and applications. The challenges and future perspectives for heparin-based materials are also discussed. Prospects are promising for heparin-containing polymeric biomaterials in diverse applications ranging from cell carriers for promoting cell differentiation to nanoparticle therapeutics for cancer treatment.

Protein Engineering by In Vivo Incorporation of Non-Natural Amino Acids: Control of Incorporation of Methionine Analogues by Methionyl-tRNA Synthetase

The incorporation of non-natural amino acids is an important strategy for engineering novel chemical and physical properties into natural and artificial proteins. The incorporation of amino acids into proteins in vivo is controlled in large part by the aminoacyl-tRNA synthetases (AARS). We have measured kinetic constants for in vitro activation of a set of methionine analogues by methionyl-tRNA synthetase (MetRS) via the ATP–PPi exchange reaction. Activation of methionine analogues in vitro correlates well with the ability of these analogues to support protein synthesis in vivo, substantiating the critical role of the AARS in controlling the incorporation of non-natural amino acids into proteins. Methionine analogues with k_(cat)/K_m values 2000-fold lower than those for methionine can support synthesis of a typical target protein (mDHFR) under standard conditions of protein expression. The kinetic constants correlate well with observed protein yields from a conventional bacterial expression host, indicating that the MetRS activity of the host can control the level of protein synthesis under certain conditions. Furthermore, increasing the MetRS activity of the bacterial host results in increased protein synthesis in media supplemented with the methionine analogues homoallylglycine and norleucine. These results suggest new strategies for incorporation of non-natural amino acids via manipulation of the AARS activity of a bacterial host.

Reversible maleimide–thiol adducts yield glutathione-sensitive poly(ethylene glycol)–heparin hydrogels

We have recently reported that retro Michael-type addition reactions can be employed for producing labile chemical linkages with tunable sensitivity to physiologically relevant reducing potentials. We reasoned that such strategies would also be useful in the design of glutathione-sensitive hydrogels for a variety of targeted delivery and tissue engineering applications. In this report, we describe hydrogels in which maleimide-functionalized low molecular weight heparin (LMWH) is crosslinked with various thiol-functionalized poly(ethylene glycol) (PEG) multi-arm star polymers. Judicious selection of the chemical identity of the thiol permits tuning of degradation via previously unstudied, but versatile chemical methods. Thiol pKa and hydrophobicity affected both the gelation and degradation of these hydrogels. Maleimide-thiol crosslinking reactions and retro Michael-type addition reactions were verified with 1H NMR during the crosslinking and degradation of hydrogels. PEGs esterified with phenylthiol derivatives, specifically 4-mercaptophenylpropionic acid or 2,2-dimethyl-3-(4-mercaptophenyl)propionic acid, induced sensitivity to glutathione as shown by a decrease in hydrogel degradation time of 4-fold and 5-fold respectively, measured via spectrophotometric quantification of LMWH. The degradation proceeded through the retro Michael-type addition of the succinimide thioether linkage, with apparent pseudo-first order reaction constants derived from oscillatory rheology experiments of 0.039 ± 0.006 h-1 and 0.031 ± 0.003 h-1. The pseudo-first order retro reaction constants were approximately an order of magnitude slower than the degradation rate constants for hydrogels crosslinked via disulfide linkages, indicating the potential use of these Michael-type addition products for reduction-mediated release and/or degradation, with increased blood stability and prolonged drug delivery timescales compared to disulfide moieties.

Identification of an expanded set of translationally active methionine analogues in Escherichia coli.

Amino acid incorporation into proteins in vivo is controlled most stringently by the aminoacyl‐tRNA synthetases. Here we report the incorporation of several new methionine analogues into protein by increasing the rate of their activation by the methionyl‐tRNA synthetase (MetRS) of Escherichia coli. cis‐Crotylglycine (4), 2‐aminoheptanoic acid (7), norvaline (8), 2‐butynylglycine (11), and allylglycine (12) will each support protein synthesis in methionine‐depleted cultures of E. coli when MetRS is overexpressed and the medium is supplemented with the analogue at millimolar concentrations. These investigations suggest important opportunities for protein engineering, as expansion of the translational apparatus toward other amino acid analogues by similar strategies should also be possible.