muramidase and maleimide

In situ encapsulation is a frequently used method to prepare hydrogels loaded with high quantities of therapeutic proteins. However, many cross-linking reactions, such as Michael-type addition or Diels-Alder (DA) reaction are not tolerant toward nucleophiles; therefore, side-reactions with proteins can occur during cross-linking. This may lead to undesired protein conjugation, activity loss and incomplete protein release. In this study, a number of polyanions, namely alginate, dextran sulfate, hyaluronic acid, heparin, and poly(acrylic acid), were screened for their capability to protect proteins during covalent cross-linking. To this end, lysozyme was incubated with furyl- and maleimide-substituted methoxy poly(ethylene glycol); different pH values were tested. The degree of PEGylation and the residual activity of lysozyme were investigated. Without polyanions, 61.1% of the total lysozyme amount was PEGylated at pH7.4; the residual activity was 20.3% of the initial activity. With the most effective polyanion (dextran sulfate), PEGylation could be completely suppressed; the residual activity was 98.4%. The protective effect of polyanions was attributed to electrostatic interactions with proteins; the "shielding" could be reversed by adding high salt concentrations. Furthermore, the protective effect was dependent on the concentration and molecular mass of the polyanion, but almost independent of the protein concentration. As a proof of concept, hydrogels were loaded with lysozyme and bevacizumab during cross-linking via DA reaction. Without polyanions, a large fraction of the protein was covalently bound to the polymer network resulting in degradation-controlled release; the residual activity of lysozyme was 50.0%. With polyanions, the protein molecules were mobile and their release was diffusion-controlled. The residual activity of lysozyme was 88.9%; the released bevacizumab was structurally intact. Polyanions can, therefore, be used as protective additive to prevent chemical protein modification during hydrogel cross-linking.

Topics: Animals; Antineoplastic Agents, Immunological; Bevacizumab; Chickens; Cross-Linking Reagents; Diffusion; Drug Delivery Systems; Drug Liberation; Hydrogels; Maleimides; Muramidase; Polyelectrolytes; Polyethylene Glycols; Polymers; Protein Stability

The compatibility of selected cross-linking reactions with lysozyme is investigated. Michael-type additions of nucleophilic amino acids to maleimide, vinyl sulfone and acrylamide groups are detected by gel electrophoresis. The degree of modification depends on the polymer and the pH. Complete modification with more than five PEG chains is observed after incubation with mPEG5k-vinyl sulfone at pH 9, whereas 96% of the protein remains unmodified after incubation with mPEG5k-acrylamide at pH 4. Incubation with mPEG5k-thiol results in thiol-disulfide exchange reactions. Hydrogel preparation is simulated by using polymer mixtures. Protein modifications are detected, which may affect the protein structure, decrease activity and bioavailability, and increase the risk for immune responses.

Topics: Animals; Chickens; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Hydrogels; Maleimides; Models, Molecular; Muramidase; Polyethylene Glycols

Single molecule Fluorescence Resonance Energy Transfer (FRET) has been widely applied to study structure, function and dynamics of complex biological systems. Labeling of proteins at specific positions with fluorescent dyes is a challenging and key step for any single molecule FRET measurement. Genetic code expansion has facilitated site specific incorporation of unnatural amino acids into proteins. These unnatural amino acid bears bioorthognal functional groups that provide opportunity to install a unique chemical handle into proteins. Propargyllysine is an unnatural amino acid which, when incorporated into a protein, can be exploited to attach commercially available fluorescent azide dyes through copper-catalyzed alkyne-azide cycloaddition click reaction (also known as click reaction). We describe here an optimized strategy to combine synthesis of propargyllysine, its genetic incorporation in the protein and click reaction to site-specifically label the protein with azide derivative of Alexa® 488. Later the protein is labeled at unique cysteine residue via maleimide coupling chemistry with acceptor Alexa® 594 dye to yield double labeled protein as required for any single molecule FRET experiments.

Topics: Amino Acid Substitution; Click Chemistry; Escherichia coli; Fenofibrate; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Genetic Vectors; Lysine; Maleimides; Muramidase; Mutagenesis, Site-Directed; Proteins; Recombinant Proteins; Staining and Labeling; Viral Proteins

Studies of protein structure and function using single-molecule fluorescence resonance energy transfer (smFRET) benefit dramatically from the ability to site-specifically label proteins with small fluorescent dyes. Genetically encoding the unnatural amino acid (UAA) p-acetylphenylalanine is an efficient way to introduce commercially available fluorescent tags with high yield and specificity. This protocol describes the expression in Escherichia coli of proteins containing this UAA in response to the amber stop codon TAG. Proteins were purified with high yield and subsequently labeled with the hydroxylamine derivative of Alexa Fluor® 488 functioning as a fluorescent donor dye. The proteins were then labeled via maleimide coupling chemistry at a unique cysteine with the acceptor dye Alexa Fluor® 594 to yield a dual-labeled protein ready for subsequent smFRET observation.

Topics: Bacteriophage T4; Binding Sites; Cysteine; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Hydroxylamine; Ketones; Maleimides; Muramidase; Oximes; Phenylalanine; Protein Engineering; Recombinant Proteins; Staining and Labeling; Transformation, Genetic

Quantitative proteomics requires novel analytical methodology to fill the gap related to absolute protein abundance in different physiological conditions. In this paper, we demonstrate a proof-of-concept study for absolute protein quantification. 1,4,7,10-Tetraazacyclododecane-1,4,7-trisacetic acid-10-maleimidoethylacetamide (MMA-DOTA) loaded with Eu was used to label lysozyme, insulin, and ribonuclease A, and they were subsequently quantified using HPLC coupled with (153)Eu species-unspecific isotope dilution inductively coupled plasma mass spectrometry (ICPMS). Labeling procedures were optimized using electrospray ionization mass spectrometry (ESI-MS) based on the labeling efficiency and specificity of the three intact proteins, which suggested that 10-fold or higher MMA-DOTA to cysteine sulphydryl rates at pH from 6.8 to 7.6 and 47 degrees C for 40 min were optimal conditions for the conjugation of the reduced-form proteins and that a 5-fold excess of Eu with respect to the DOTA present in the MMA-DOTA-conjugated proteins and pH 5.8 are optimal for Eu labeling. Subsequently, these three MMA-DOTA-Eu-labeled proteins were digested with trypsin, and the tryptic peptides were quantified via HPLC coupled with (153)Eu species-unspecific isotope dilution ICPMS. The results for the protein studied indicated that not only could 100% digestion efficiency not be achieved but also the resulting peptides needed a chromatographic separation at higher resolution. On the other hand, the labeled intact proteins were quantified without tryptic digestion. The average recovery was found to be 97.9% in six independent experiments, and the precision was evaluated to be 5.8% at the 10 pmol L(-1) level. The detection limits (3sigma) were determined to be 0.819, 1.638, and 0.819 fmol for lysozyme, the A chain of insulin, and ribonuclease A, respectively, using ICPMS with a normal concentric pneumatic nebulizer. These results demonstrated that high-quality absolute protein quantification could be achieved through labeling the intact proteins but not the tryptic peptides, implying that intact proteins may be more feasible and practical targets than tryptic peptides for ICPMS-based absolute protein quantification.

Topics: Amino Acid Sequence; Animals; Cattle; Chelating Agents; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Europium; Hydrogen-Ion Concentration; Insulin; Kinetics; Maleimides; Mass Spectrometry; Molecular Sequence Data; Muramidase; Organometallic Compounds; Oxidation-Reduction; Peptide Fragments; Proteins; Ribonuclease, Pancreatic; Staining and Labeling; Substrate Specificity; Temperature; Trypsin

Maleimide end functionalized polymers for site-selective conjugation to free cysteines of proteins were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. A furan-protected maleimide chain transfer agent (CTA) was employed in the RAFT polymerization of poly(ethylene glycol) methyl ether acrylate (PEGA). Gel permeation chromatography (GPC) with laser light scattering detection indicated that 20,000 and 39,000 Da polyPEGA had been made with polydispersity indices of 1.25 and 1.36, respectively. The maleimide group on the polymer chain end was exposed by heating the poly(PEGA)s for 4 h. The deprotection efficiency was estimated to be 80 and 60% for poly(PEGA)(20 kDa) and poly(PEGA)(39 kDa), respectively. Maleimide-poly(PEGA)s were conjugated to V131C T4 lysozyme (T4L), and the resultant polymer-protein conjugates were characterized by size exclusion chromatography and gel electrophoresis.

Topics: Chromatography, Gel; Cysteine; Electrophoresis, Polyacrylamide Gel; Maleimides; Molecular Weight; Muramidase; Polyethylene Glycols; Polymerization; Proteins