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 cell's 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.

This review provides insight into emerging degradable and cell-compatible hydrogels for understanding and modulating cell behavior for various bioengineering applications.

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 a Michael type addition of a thiol to N-ethylmaleimide (NEM), generally accepted as stable, can in fact undergo 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 (1H 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 20–80 hrs, with extents of conversion from 20–90% for MPA and N-acetylcysteine conjugates. Ring-opened the resultant succinimide thioether as well as any MP adduct did not show retro and exchange reactions. The kinetics of the retro reactions can be modulated by the Michael donor’s reactivity; therefore the degradation of maleimide-thiol adducts could be tuned for controlled release of drugs or degradation of materials at timescales 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 sub-cellular trafficking.

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, as well as 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.

A set of eight methionine analogues was assayed for translational activity in Escherichia coli. Norvaline and norleucine, which are commercially available, were assayed along with 2-amino-5-hexenoic acid (2), 2-amino-5-hexynoic acid (3), cis-2-amino-4-hexenoic acid (4), trans-2-amino-4-hexenoic acid (5), 6,6,6-trifluoro-2-aminohexanoic acid (6), and 2-aminoheptanoic acid (7), each of which was prepared by alkylation of diethyl acetamidomalonate with the appropriate tosylate, followed by hydrolysis. The E. coli methionine auxotroph CAG18491, transformed with plasmids pREP4 and pQE15, was used as the expression host, and translational activity was assayed by determination of the capacity of the analogue to support synthesis of the test protein dihydrofolate reductase (DHFR) in the absence of added methionine. The importance of amino acid side chain length was illustrated by the fact that neither norvaline (8) nor 7 showed translational activity, in contrast to norleucine (9), which does support protein synthesis under the assay conditions. The internal alkene functions of 4 and 5 prevented incorporation of these analogues into test protein, and the fluorinated analogue 6 yielded no evidence of translational activity. The terminally unsaturated compounds 2 and 3, however, proved to be excellent methionine surrogates: 1 H NMR spectroscopy, amino acid analysis, and N-terminal sequencing indicated ∼85% substitution of methionine by 2, while 3 showed 90-100% replacement. Both analogues also function efficiently in the initiation step of protein synthesis, as shown by their near-quantitative occupancy of the N-terminal amino acid site in DHFR. Enzyme kinetics assays were conducted to determine the rate of activation of each of the methionine analogues by methionyl tRNA synthetase (MetRS); results of the in vitro assays corroborate the in vivo incorporation results, suggesting that success or failure of analogue incorporation in vivo is controlled by MetRS.

The noncovalent assembly of polymeric materials via specific molecular recognition interactions has become increasingly prominent in the production of responsive, reversible, and injectable hydrogels. While protein-protein interactions predominantly have been used for such assembly, 1-4 the use of extracellular matrix (ECM)-derived polysaccharide-peptide interactions has been recently emerging as an assembly approach. 5-9 The interactions of proteins and glycosaminoglycans are critical in mediating the multiple responses of the ECM, and materials in which assembly and mechanical properties are controlled by such molecular recognition events may be useful for biologically directed targeted delivery. Accordingly, the ability of heparin and related glycosaminoglycans to sequester and stabilize growth factors has been exploited in the production of surfaces and covalently crosslinked hydrogels that can mediate cell proliferation and migration, angiogenesis, and wound healing. 10-14 In particular, the controlled delivery of VEGF (vascular endothelial growth factor) and select other growth factors from polymeric matrices has shown potential for use in vascular therapies. 12,15,16 Noncovalently assembled matrices also have similar potential use in this regard. 5-9Here, we report the assembly, rheological properties, and targeted delivery/erosion profiles of noncovalently associated hydrogel networks produced via the interaction of a low-molecularweight heparin-modified star polymer (PEG-LMWH) and a dimeric, heparin-binding growth factor (VEGF). 17 Given that overexpression of VEGF and other growth factor receptors plays a key role in both normal healing and pathological conditions, 18-20 we reasoned that hydrogels assembled via such strategies may provide unique opportunities for stimuli-responsive delivery and erosion via biologically relevant, ligand-exchange mechanisms. A schematic of the assembly and erosion strategy is illustrated in Figure 1. We have focused on LMWH in these investigations in order to maximize intermolecular dimeric growth factor/LMWH binding over intramolecular binding (see Supporting Information). The PEGLMWH employed in these investigations was produced via Michael addition of thiol-terminated four-arm star PEG to maleimidefunctionalized LMWH. 7 NMR characterization of the purified product indicated at least 75% functionalization of PEG-LMWH, indicating that it is of sufficiently high functionality (f > 2) to permit formation of crosslinked networks upon interaction with VEGF (f = 2). The VEGF was expressed from E. coli and purified via heparin-affinity chromatography as previously described (Supporting Information). 16Correspondence to: Kristi L. Kiick. Hydrogels were formed via the mixing of homogeneous, lowviscosity solutions of each component in phosphate buffered saline (PBS). The PEG-LMWH solution was vortexed to ensure homogeneity. Addition of a solution of VEGF (5 μL, 2 mg/mL) to a solution of PEG-LMWH (5 μL, 80 mg/mL) immediately resulted in the formation of a self-supporting, viscoel...

The production of complex, yet well defined materials offers many opportunities in regenerative medicine, in which the mechanical and biological properties of the matrix must meet stringent requirements. Here we report the recombinant production of modular polypeptidic materials, based on the highly resilient protein resilin, which are equipped with multiple biologically active domains. The recombinant materials exhibit useful mechanical and cell adhesion behavior.

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.