Terminal Cysteine Residue Research Articles

A universal method for the purification of C2H2 zinc finger arrays.

Zinc fingers (ZFs) are compact, modular, sequence-specific polynucleotide-binding domains uniquely suited for use as DNA probes and for the targeted delivery of effector domains for purposes such as gene regulation and editing. Despite recent advances in both the design and application of ZF-containing proteins, there is still a lack of a general method for their expression and purification. Here we describe a simple method, involving two chromatographic steps, for the production of homogeneous, functional ZF proteins in high yield (one milligram per liter of bacterial culture), and we demonstrate the generality of this method by applying it to a diverse set of eight C2H2-type ZF proteins. By incorporating a surface-exposed terminal cysteine residue that enables site-specific conjugation with maleimide-activated fluorophores, we confirm the suitability of these probes for in situ labeling of specific DNA sequences in human cells.

Light-triggered AND logic tetrapeptide dynamic covalent assembly

We demonstrate light-triggered dynamic covalent assembly of a linear short tetrapeptide containing two terminal cysteine residues in an AND logic manner. A photobase generator is introduced to accomplish light-mediated pH regulation to increase the reduction potential of thiols in the tetrapeptide, which activates its oxidative polymerization through disulfide bonds. Interestingly, it is elucidated that under light irradiation, mere co-existence of photobase generator and the oxidizing agent permits the polymerization performance of this tetrapeptide. Hence, a light-triggered AND logic dynamic covalent assembly of a tetrapeptide is achieved. Further, upon redox response, the reversible aggregation and disaggregation can be transformed for numerous times due to the dynamic covalent feature of disulfide bond. As a comparison, no assembly occurs for a short peptide containing one terminal cysteine residue under the same stimuli condition. This work offers a new approach to remotely control programmable molecular assembly of short linear peptides based on dynamic covalent bond, holding great potential in wide bioapplications.

Grafting Strategies of Oxidation-Prone Coiled-Coil Peptides for Protein Capture in Bioassays: Impact of Orientation and the Oxidation State.

Many biomedical and biosensing applications require functionalization of surfaces with proteins. To this end, the E/K coiled-coil peptide heterodimeric system has been shown to be advantageous. First, Kcoil peptides are covalently grafted onto a given surface. Ecoil-tagged proteins can then be non-covalently captured via a specific interaction with their Kcoil partners. Previously, oriented Kcoil grafting was achieved via thiol coupling, using a unique Kcoil with a terminal cysteine residue. However, cysteine-terminated Kcoil peptides are hard to produce, purify, and oxidize during storage. Indeed, they tend to homodimerize and form disulfide bonds via oxidation of their terminal thiol group, making it impossible to later graft them on thiol-reactive surfaces. Kcoil peptides also contain multiple free amine groups, available for covalent coupling through carbodiimide chemistry. Grafting Kcoil peptides on surfaces via amine coupling would thus guarantee their immobilization regardless of their terminal cysteine's oxidation state, at the expense of the control over their orientation. In this work, we compare Kcoil grafting strategies for the subsequent capture of Ecoil-tagged proteins, for applications such as surface plasmon resonance (SPR) biosensing and cell culture onto protein-decorated substrates. We compare the "classic" thiol coupling of cysteine-terminated Kcoil peptides to the amine coupling of (i) monomeric Kcoil and (ii) dimeric Kcoil-Kcoil linked by a disulfide bond. We have observed that SPR biosensing performances relying on captured Ecoil-tagged proteins were similar for amine-coupled dimeric Kcoil-Kcoil and thiol-coupled Kcoil peptides, at the expense of higher Ecoil-tagged protein consumption. For cell culture applications, Ecoil-tagged growth factors captured on amine-coupled monomeric Kcoil signaled through cell receptors similarly to those captured on thiol-coupled Kcoil peptides. Altogether, while oriented thiol coupling of cysteine-terminated Kcoil peptides remains the most reliable and versatile platform for Ecoil-tagged protein capture, amine coupling of Kcoil peptides, either monomeric or dimerized through a cysteine bond, can offer a good alternative when the challenges and costs associated with the production of monomeric cysteine-tagged Kcoil are too dissuasive for the application.

The Fully Oxidized State of the Glutamate Coordinated O2-Tolerant [NiFe]-Hydrogenase Shows a Ni(III)/Fe(III) Open-Shell Singlet Ground State.

The oxygen tolerance of the [NiFe]-hydrogenase from H. thermoluteolus was recently assigned to originate from an unusual coordination sphere of the active site nickel atom (Shomura et al. Science 2017, 357, 928-932, 10.1126/science.aan4497). In the oxidized state, a terminal cysteine residue is displaced by a bidentate coordinating nearby Glu32 and thus moves to occupy a third μ-cysteine bridging position. Spectral features of the oxidized state were assigned to originate from a closed-shell Ni(IV)/Fe(II) state (Kulka-Peschke et al. J. Am. Chem. Soc. 2022, 144, 17022-17032, 10.1021/jacs.2c06400). Such a high-valent nickel oxidation state is unprecedented in biological systems. The spectral properties and the coordination sphere of that [NiFe]-hydrogenase can, however, also be rationalized by an energetically lower broken-symmetry Ni(III)/Fe(III) state of the active site which was not considered. In this open-shell singlet, the ligand-mediated antiferromagnetic spin-coupling leads to an overall S = 0 spin state with evenly distributed spin densities over the metal atoms. Experiments are suggested that may clarify the final assignment of redox states.

Functionalization of polymeric nanoparticles with targeting VNAR ligands using vinyl sulfone conjugation.

Actively targeted drug loaded nanoparticles represent an exciting new form of therapeutics for cancer and other diseases. These formulations are complex and in order to realize their ultimate potential, optimization of their preparation is required. In this current study, we have examined the conjugation of a model targeting ligand, conjugated in a site-specific manner using a vinyl sulfone coupling approach. A disulfide-functionalized poly(L-lactide)-b-poly(oligo(ethylene glycol) methacrylate)-stat-(bis(2-methacryloyl)oxyethyl disulfide) (PLA-b-P(OEGMA-stat-DSDMA)) diblock copolymer was synthesized by simultaneous ring opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) polymerization. Subsequently, the disulfide bonds of the polymer were reduced to thiols and divinyl sulfone was attached to the polymer using thiol-ene chemistry to produce the vinyl sulfone (VS)-functionalized PLA-b-P(OEGMA-stat-VSTEMA) amphiphilic block copolymer. Single emulsion - solvent evaporation was employed using a blend of this polymer with poly(D,L-lactide-co-glycolide) (PLGA) to produce VS-functionalized polymeric nanoparticles. The ability of these novel nanoparticles to attach ligands was then exemplified using a single domain variable new antigen receptor (VNAR) with a free carboxyl terminal cysteine residue. The resulting VNAR-functionalized nanoparticles were found to maintain specific affinity to their cognate antigen (DLL4) for at least 72 h at 4 °C. The simplicity of the degradable amphiphilic block copolymer synthesis and the efficiency of VNAR conjugation to the VS-functionalized nanoparticles show the potential of this platform for therapeutic development.

Peptide Inhibitors of the Interaction of the SARS-CoV-2 Receptor-Binding Domain with the ACE2 Cell Receptor.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin-converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22–23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which terminal cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of each precursor peptide molecule. The precursors h1 and h2 mimic amino acid sequences of α1- and α2-helices of the ACE2 extracellular peptidase domain, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their constituent peptides with RBD (particularly in dependence of the middle and terminal methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified to 95–97% purity, and characterized by HPLC and MALDI-TOF mass spectrometry. Binding of these peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. The original RBD of the Chinese variant bound to three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant. The antiviral activity of the proposed peptides was evaluated in Vero cell line.

Surface Ligand Valency and Immunoliposome Binding: when More Is Not Always Better.

Nano-drug delivery systems are designed to contain surface ligands including antibodies for "active targeting". The number of ligands on each nanoparticle, known as the valency, is considered a critical determinant of the "targeting" property. We sought to understand the correlation between valency and binding properties using antibody conjugated liposomes, i.e. immunoliposomes (ILs), as the model. Anti-CD3 Fab containing a terminal cysteine residue were conjugated to DSPE-PEG-maleimide and incubated with preformed liposomes at 60°C. The un-incorporated antibodies were removed and the obtained ILs were characterized to contain in average 2-22 copies of anti-CD3 Fabs per liposome. The Biolayer Interferometry (BLI) probe surface was coated with various densities of CD3 epsilon&delta heterodimer (CD3D/E) to imitate different CD3 expression levels on target cells. The inference wavelength shifts upon anti-CD3 liposome binding were monitored and analyzed. The data indicated ILs may bind either monovalently or multivalently, determined mainly by the surface ligand density rather than the ILs antibody valency. The ILs valency indeed correlated with the dissociation rate constant (Koff), but not with the association rate constant (Kon). Their binding capabilities also did not necessarily increase with the surface anti-CD3 valency. We proposed a model for understanding the binding properties of ILs with different ligand valencies. The binding mode may change when the targeted surfaces had different antigen densities. The model should be important for the designing and optimization of active targeting drug delivery systems to fit different applications.

SARS-CoV-2 Peptide Bioconjugates Designed for Antibody Diagnostics.

In the near future, the increase in the number of required tests for COVID-19 antibodies is expected to be many hundreds of millions. Obviously, this will be done using a variety of analytical methods and using different antigens, including peptides. In this work, we compare three method variations for detecting specific immunoglobulins directed against peptides of approximately 15-aa of the SARS-CoV-2 spike protein. These linear peptide epitopes were selected using antigenicity algorithms, and were synthesized with an additional terminal cysteine residue for their bioconjugation. In two of the methods, constructs were prepared where the peptide (F, function) is attached to a negatively charged hydrophilic spacer (S) linked to a dioleoylphosphatidyl ethanolamine residue (L, lipid) to create a function-spacer-lipid construct (FSL). These FSLs were easily and controllably incorporated into erythrocytes for serologic testing or in a lipid bilayer deposited on a polystyrene microplate for use in an enzyme immunoassays (EIA). The third method, also an EIA, used polyacrylamide conjugated peptides (peptide-PAA) prepared by controlled condensation of the cysteine residue of the peptide with the maleimide-derived PAA polymer which were immobilized on polystyrene microplates by physisorption of the polymer. In this work, we describe the synthesis of the PAA and FSL peptide bioconjugates, design of test systems, and comparison of the bioassays results, and discuss potential reasons for higher performance of the FSL conjugates, particularly in the erythrocyte-based serologic assay.

Combining Metabolic Alkyne Labeling and Click Chemistry for Secretome Analysis of Serum‐Containing Conditioned Medium†

Main observation and conclusionBioorthogonal click chemistry has emerged as a powerful tool for the specific modification of proteins in complex mixtures. Metabolic labeling of proteins with azide followed by the copper‐catalyzed azide−alkyne cycloaddition (CuAAC) with alkyne‐based affinity probes/beads is widely applied to study protein turnover and post‐translational modifications (PTMs). However, it has long been known that the alkyne‐based enrichment of high concentration protein samples (e.g., serum) is not sufficient to remove high abundant contaminating proteins. Herein, we demonstrate that the protein contamination is mainly caused by thiol‐yne addition between terminal alkyne and cysteine residue. Furthermore, we report that azide‐based enrichment combined with metabolic labeling with alkyne can significantly reduce contamination and improve the enrichment efficiency of secreted proteins in serum‐containing conditioned medium.

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Disulfide bond of Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin is essential to maintain the ADP-ribosylating and vacuolating activities.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalised children in United States and worldwide. Community-acquired respiratory distress syndrome (CARDS) toxin is a key virulence determinant of M. pneumoniae. The N-terminus of CARDS toxin exhibits ADP-ribosyltransferase (ADPRT) activity, and the C-terminus possesses binding and vacuolating activities. Thiol-trapping experiments of wild-type (WT) and cysteine-to-serine-mutated CARDS toxins with alkylating agents identified disulfide bond formation at the amino terminal cysteine residues C230 and C247. Compared with WT and other mutant toxins, C247S was unstable and unusable for comparative studies. Although there were no significant variations in binding, entry, and retrograde trafficking patterns of WT and mutated toxins, C230S did not elicit vacuole formation in intoxicated cells. In addition, the ADPRT domain of C230S was more sensitive to all tested proteases when compared with WT toxin. Despite its in vitro ADPRT activity, the reduction of C230S CARDS toxin-mediated ADPRT activity-associated IL-1β production in U937 cells and the recovery of vacuolating activity in the protease-released carboxy region of C230S indicated that the disulfide bond was essential not only to maintain the conformational stability of CARDS toxin but also to properly execute its cytopathic effects.

30 - Drosophila Methionine sulfoxide reductase A is not a methionine oxidase

Methionine sulfoxide reductase A (MsrA) stereospecifically catalyzes the reduction of S-methionine sulfoxide to methionine and is important in defense against oxidative stress. Recently, we reported that mammalian methionine sulfoxide reductase A stereospecifically and selectively oxidizes Met77 in calcium-bound calmodulin and can fully reduce it as well. The control mechanism that prevents futile cycling is hypothesized to be through interaction with a postulated regulatory protein. Thus, cyclic oxidation and reduction of methionines in proteins by MsrA could function as a redox-based mechanism of cellular regulation. Our aim in this study was to elucidate the physiological significance of methionine sulfoxide reductase A mediated reversible oxidation of calmodulin Met77 in Drosophila. However, we found that Drosophila MsrA, unlike its mammalian counterpart, is not a methionine oxidase. This led us to explore the mechanistic details of the enzyme. Using a double alkylation approach with HPLC-mass spectrometric sequencing, we found that the active site cysteine residue in Drosophila MsrA becomes locked in a disulfide bond with the terminal cysteine residue of the protein and thus cannot mediate oxidation. A mutant Drosophila MsrA lacking the two C-terminal cysteine residues also lacked oxidase activity, despite not being able to form a disulfide bond with the active site cysteine.

Abstract 1879: Structural and biochemical characterization of farnesylated and methylated KRAS-membrane interactions

Abstract KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed in several enzymatic steps that results in the addition of a farnesyl and a methyl group to the terminal cysteine residue. Plasma membrane localization of KRAS4b requires this processing along with a polybasic stretch of lysines within the hypervariable region. KRAS4b can bind to RAF-RBD in solution but membrane bound KRAS4b is required for RAF-kinase activation. In order to determine why membrane interaction of KRAS4b is required for MAPK signal transduction we under took a structural and biochemical analysis of KRAS4b-membrane interactions. We have developed methods using an engineered baculovirus for the insect cell production of farnesylated and methylated KRAS4b (KRAS4b-FME) and this material was used in these studies. We have demonstrated that the stable interaction of KRAS4b-FME to lipid Nanodiscs and tethered lipid bilayers is dependent on the presence of anionic phospholipids. Using analytical ultracentrifugation we have evaluated the binding stoichiometry of KRAS4b-FME with Nanodiscs. In addition we have used neutron reflectivity to determine the orientation of KRAS4b-FME on tethered lipid bilayers. Citation Format: Que Van, William K. Gillette, Dominic Esposito, Rodolfo Ghirlando, Frank Heinrich, Andrew G. Stephen. Structural and biochemical characterization of farnesylated and methylated KRAS-membrane interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1879.

Abstract 2168: Optimizing doxorubicin derivatives delivery using temperature sensitive biopolymers in multidrug resistant breast cancer cells

Abstract The anticancer agent doxorubicin is an anthracycline compound that shows high potency in treating cancer, and it is one of the most widely used chemotherapeutics. However, efficiency of doxorubicin treatment is limited by low blood plasma solubility, poor blood pharmacokinetics, and non-selective cell killing that results in serious toxicity to healthy tissues. Additionally, cancer cells often develop drug resistance, which is a significant limiting factor to the drug's effectiveness. Motivated by these problems, we have designed a drug delivery system that can specifically deliver drug to the tumor site while overcoming doxorubicin resistance in breast cancer cell lines. This drug delivery system consists of: ELP - Elastin like polypeptide, CPP - Cell penetrating peptide, a cleavable linker – to enable doxorubicin release in the targeted low pH environment, and a derivative of the anticancer agent Doxorubicin (modified by a 6-maleimidocaproyl moiety for conjugation to a terminal cysteine residue on ELP). ELP is thermally responsive and improves the complex's pharmacokinetics by prolonging its clearance rate while the CPP mediates cellular uptake of large macromolecules. The linker is an acid sensitive amino acid sequence (Gly-Phe-Leu-Gly) that serves as a substrate for lysosomal enzymes. In this study, we compared cytotoxicity of a cleavable (cDox) and non-cleavable (ncDox) doxorubicin derivative delivered by ELP biopolymer in the breast cancer cell lines MCF-7 and MCF7/ADR (doxorubicin resistant cell line). We showed that cDox had two fold higher cytotoxicity than ncDox in both cell lines. When ncDox, however, was conjugated to the ELP biopolymer containing a lysosomally degradable GFLG spacer, the drug delivery construct was equally cytotoxic to both sensitive and resistant cell lines, indicating that the construct delivers doxorubicin into cells by a mechanism that bypasses doxorubicin resistance. Confocal fluorescence microscopy showed that after two hours of exposure to the doxorubicin derivatives, cDox was predominantly localized in the nucleus in both cell lines. However, ncDox was localized in the plasma membrane and cytosol. Intracellular doxorubicin accumulation examined by flow cytometry indicated 3 fold higher uptake of ELP-cDox in sensitive MCF 7 cells compared to resistant MCF7 ADR cells. Cellular uptake of ELP-cDox was further enhanced two fold when conjugated with CPP. In conclusion, our current results indicate that ELP-doxorubicin conjugates may successfully overcome drug resistance in breast cancer cells, providing a promising approach for the use of chemotherapeutic agents such as doxorubicin in patients with drug resistant breast cancers. Citation Format: Sonja Dragojevic, Jung Su Ryu, Felix Kratz, Drazen Raucher. Optimizing doxorubicin derivatives delivery using temperature sensitive biopolymers in multidrug resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2168.

A novel 2-cyanobenzothiazole-based (18)F prosthetic group for conjugation to 1,2-aminothiol-bearing targeting vectors.

In a bid to find an efficient means to radiolabel biomolecules under mild conditions for PET imaging, a bifunctional (18)F prosthetic molecule has been developed. The compound, dubbed [(18)F]FPyPEGCBT, consists of a 2-substituted pyridine moiety for [(18)F]F(-) incorporation and a 2-cyanobenzothiazole moiety for coupling to terminal cysteine residues. The two functionalities are separated by a mini-PEG chain. [(18)F]FPyPEGCBT could be prepared from its corresponding 2-trimethylammonium triflate precursor (100 °C, 15 min, MeCN) in preparative yields of 11% ± 2 (decay corrected, n = 3) after HPLC purification. However, because the primary radiochemical impurity of the fluorination reaction will not interact with 1,2-aminothiol functionalities, the (18)F prosthetic could be prepared for bioconjugation reactions by way of partial purification on a molecularly imprinted polymer solid-phase extraction cartridge. [(18)F]FPyPEGCBT was used to (18)F-label a cyclo-(RGDfK) analogue which was modified with a terminal cysteine residue (TCEP·HCl, DIPEA, 30 min, 43 °C, DMF). Final decay-corrected yields of (18)F peptide were 7% ± 1 (n = 9) from end-of-bombardment. This novel integrin-imaging agent is currently being studied in murine models of cancer. We argue that [(18)F]FPyPEGCBT holds significant promise owing to its straightforward preparation, 'click'-like ease of use, and hydrophilic character. Indeed, the water-tolerant radio-bioconjugation protocol reported herein requires only one HPLC step for (18)F peptide purification and can be carried out remotely using a single automated synthesis unit over 124-132 min.

Self-Assembling Multifunctional Peptide Dimers for Gene Delivery Systems

Self-assembling multifunctional peptide was designed for gene delivery systems. The multifunctional peptide (MP) consists of cellular penetrating peptide moiety (R8), matrix metalloproteinase-2 (MMP-2) specific sequence (GPLGV), pH-responsive moiety (H5), and hydrophobic moiety (palmitic acid) (CR8GPLGVH5-Pal). MP was oxidized to form multifunctional peptide dimer (MPD) by DMSO oxidation of thiols in terminal cysteine residues. MPD could condense pDNA successfully at a weight ratio of 5. MPD itself could self-assemble into submicron micelle particles via hydrophobic interaction, of which critical micelle concentration is about 0.01 mM. MPD showed concentration-dependent but low cytotoxicity in comparison with PEI25k. MPD polyplexes showed low transfection efficiency in HEK293 cells expressing low level of MMP-2 but high transfection efficiency in A549 and C2C12 cells expressing high level of MMP-2, meaning the enhanced transfection efficiency probably due to MMP-induced structural change of polyplexes. Bafilomycin A1-treated transfection results suggest that the transfection of MPD is mediated via endosomal escape by endosome buffering ability. These results show the potential of MPD for MMP-2 targeted gene delivery systems due to its multifunctionality.

Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform

Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

2-Cyanobenzothiazole (CBT) condensation for site-specific labeling of proteins at the terminal cysteine residues.

Site specificity is pivotal in obtaining homogeneously labeled proteins without batch-to-batch variations. More importantly, precisely controlled modification at specific sites avoids potential pitfalls that could otherwise interfere with protein folding, structure, and function. Inspired by the chemical synthesis of D-luciferin, we have developed an efficient strategy (second-order rate constant k 2 = 9.2 M(-1) s(-1)) for labeling of proteins containing 1,2-aminothiol via reaction with 2-cyanobenzothiazole (CBT). In addition, the CBT condensation enjoys the convenience of protein engineering, as production of N-terminal cysteine-containing proteins has been well developed for native chemical ligation. This protocol describes the preparation of Renilla luciferase (rLuc) with 1,2-aminothiol at either its N- or C-terminus, and site-specific labeling of rLuc with fluorescein or (18)F via CBT condensation.

Development and in vitro assessment of enzymatically-responsive poly(ethylene glycol) hydrogels for the delivery of therapeutic peptides

Despite the recent expansion of peptide drugs, delivery remains a challenge due to poor localization and rapid clearance. Therefore, a hydrogel-based platform technology was developed to control and sustain peptide drug release via matrix metalloproteinase (MMP) activity. Specifically, hydrogels were composed of poly(ethylene glycol) and peptide drugs flanked by MMP substrates and terminal cysteine residues as crosslinkers. First, peptide drug bioactivity was investigated in expected released forms (e.g., with MMP substrate residues) in vitro prior to incorporation into hydrogels. Three peptides (Qk (from Vascular Endothelial Growth Factor), SPARC113, and SPARC118 (from Secreted Protein Acidic and Rich in Cysteine)) retained bioactivity and were used as hydrogel crosslinkers in full MMP degradable forms. Upon treatment with MMP2, hydrogels containing Qk, SPARC113, and SPARC118 degraded in 6.7, 6, and 1 days, and released 5, 8, and, 19% of peptide, respectively. Further investigation revealed peptide drug size controlled hydrogel swelling and degradation rate, while hydrophobicity impacted peptide release. Additionally, Qk, SPARC113, and SPARC118 releasing hydrogels increased endothelial cell tube formation 3.1, 1.7, and 2.8-fold, respectively. While pro-angiogenic peptides were the focus of this study, the design parameters detailed allow for adaptation of hydrogels to control peptide release for a variety of therapeutic applications.

ChemInform Abstract: Traceless Chemical Ligation from S‐, O‐, and N‐Acyl Isopeptides

AbstractReview: 54 refs.