7 research outputs found
One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester
<p>Abstract</p> <p>Background</p> <p>Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.</p> <p>Results</p> <p>A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation.</p> <p>Conclusion</p> <p>An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.</p
Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester
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75302.pdf (publisher's version ) (Open Access)9 p
Peptides and proteins in dendritic assemblies
Multiple, simultaneous interactions are often used in biology to enhance the affinity and specificity of binding, an effect referred to as multivalency. This multivalency can be mimicked by anchoring multiple peptides and proteins onto synthetic dendritic scaffolds. The aim of this research was to develop general methods to obtain well-defined protein and peptide assemblies, and to study multivalent interactions of these assemblies in a controlled fashion. In Chapter 2, a general synthetic strategy is described to obtain multivalent peptides and proteins using native chemical ligation. Different generations of poly(propylene imine) dendrimers were functionalized with N-terminal cysteine residues to allow the native chemical ligation reaction with C-terminal thioesters. Ligation of a peptide thioester with cysteine-functionalized dendrimers yielded multivalent peptide dendrimers of different generations with 4 to 16 peptides per dendrimer. This native chemical ligation strategy was expanded to recombinant proteins by employing intein-mediated protein expression and purification to obtain fluorescent proteins modified with a C-terminal thioester. Native chemical ligation of GFP-MESNA with a cysteine-modified dendrimer followed by ligation with peptide thioesters gives access to novel hybrid peptide-protein dendrimers. Ligation of 4 equivalents of GFP-MESNA with the cysteine-modified dendrimer yielded a branched, multivalent protein tetramer. Size exclusion chromatography combined with mass spectrometry proved to be an invaluable tool to study these complex bio-macromolecules. The use of surface plasmon resonance (SPR) biosensors enables real-time detection and monitoring of biomolecular binding events. Chapter 3 describes a chemoselective immobilization strategy for Biacore SPR sensor chips, based on native chemical ligation. First, a thioproline was introduced on the surface, which could be deprotected using mild conditions to an N-terminal cysteine residue. A streptavidin-binding peptide was immobilized via its C-terminus onto the biosensor chip, and subsequent binding experiments with streptavidin showed specific and reproducible binding to the peptide surface. Short ligation steps of peptide thioester were alternated with streptavidin binding experiments on a single chip. This provided an increased peptide loading after each ligation step, yielding enhanced protein-binding capacity. As an example of a recombinant protein, green fluorescent protein (GFP) was immobilized on the biosensor surface. Again, binding experiments with an antibody directed against GFP showed the specificity and robustness of the coupling strategy. The immobilization of S-peptide via native chemical ligation was used to illustrate the possibility of obtaining kinetic information from the specific interaction between S-peptide and S-protein. The presented approach allows for efficient immobilization of both recombinant proteins and synthetic peptides with high control over the degree of functionalization of the surface. In Chapter 4, native chemical ligation was used to synthesize multivalent peptides based on a streptavidin-binding peptide sequence derived from phage display. The synthetic multivalent scaffolds were used to mimic the multivalent character of the peptides on the head of a phage, without the presence of the phagemid coat proteins or genetic information. Peptides with different valency (from 1–4 copies per scaffold) and spacing were prepared and their affinity for streptavidin was measured using SPR. All multivalent peptides showed a significant increase in affinity compared to their monovalent counterpart and a binding model was used to describe the multivalent effect in a quantitative manner. However, the peptide dendrimers still showed considerably lower affinity than the streptavidin-binding phage. Possible reasons for this difference are discussed in this Chapter, as well as suggestions for further improvement of this dendrimer display by optimization of both scaffold rigidity and spacing of ligands. Although the covalent conjugation strategy described in Chapter 2 allowed the synthesis of tetravalent protein dendrimers of 110 kDa, non-covalent synthetic strategies are required for the development of even more complex protein assemblies. Chapter 5 explores the suitability of using the S-peptide–S-protein interaction to obtain well-defined, stable protein dendrimers. Association of S-peptide and S-protein results in the formation of an active enzyme, ribonuclease S, whereas neither fragment alone displays any enzyme activity. Native chemical ligation was used to couple four S-peptides via their C-terminal thioester to a cysteine-functionalized dendritic scaffold to yield a tetravalent S-peptide dendrimer. A fully functional ribonuclease S tetramer was prepared by addition of four equivalents of S-protein. Different biophysical techniques (ITC, SPR and mass spectrometry), and a fluorescent enzyme activity assay were used to quantify complex formation. For the non-covalent synthesis of more complex dendritic architectures, S-protein building blocks are required. Thioester-modified RNase A was obtained via recombinant expression as a precursor in the synthesis of multivalent S-protein assemblies. This noncovalent synthetic strategy based on ribonuclease S can be used to synthesize semisynthetic protein assemblies such as supramolecular polymers, gels and polymer networks with high control of structural organization, and may find applications in nanomedicine or functional biomaterial
From Phage Display to Dendrimer Display: Insights into Multivalent Binding
Phage display is widely used for the selection of target-specific peptide sequences. Presentation of phage peptides on a multivalent platform can be used to (partially) restore the binding affinity. Here, we present a detailed analysis of the effects of valency, linker choice, and receptor density on binding affinity of a multivalent architecture, using streptavidin (SA) as model multivalent receptor. For surfaces with low receptor densities, the SA binding affinity of multivalent dendritic phage peptide constructs increases over 2 orders of magnitude over the monovalent species (e.g., Kd,mono = 120 μM vs Kd,tetra = 1 μM), consistent with previous work. However, the affinity of the SA-binding phage presenting the exact same peptides was 16 pM when dense receptor surfaces used for initial phage display were used in assays. The phage affinity for SA-coated surfaces weakens severely toward the nanomolar regime when surface density of SA is decreased. A similarly strong dependence in this respect was observed for dendritic phage analogues. When presented with a dense SA-coated surface, dendrimer display affords up to a 104-fold gain in affinity over the monovalent peptide. The interplay between ligand valency and receptor density is a fundamental aspect of multivalent targeting strategies in biological systems. The perspective offered here suggests that in vivo targeting schemes might best be served to conduct ligand selection under physiologically relevant receptor density surfaces, either by controlling the receptor density placed at the selection surface or by using more biologically relevant intact cells and tissues
Multivalent peptide and protein dendrimers using native chemical ligation
A wide variety of well-defined multivalent peptides and proteins can be made by conjugating synthetic peptides and recombinantly expressed proteins to cysteine-functionalized dendrimers using native chemical ligation (see picture). This modular approach provides access to dendrimers that are attractive both for understanding fundamental issues of multivalency in biological interactions as well as for biomedical applications
Synthesis of DOTA-conjugated multivalent cyclic-RGD peptide dendrimers via 1,3-dipolar cycloaddition and their biological evaluation: implications for tumor targeting and tumor imaging purposes.
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52270.pdf (publisher's version ) (Open Access)This report describes the design and synthesis of a series of alpha(V)beta(3) integrin-directed monomeric, dimeric and tetrameric cyclo[Arg-Gly-Asp-d-Phe-Lys] dendrimers using "click chemistry". It was found that the unprotected N-epsilon-azido derivative of cyclo[Arg-Gly-Asp-d-Phe-Lys] underwent a highly chemoselective conjugation to amino acid-based dendrimers bearing terminal alkynes using a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition. The alpha(V)beta(3) binding characteristics of the dendrimers were determined in vitro and their in vivoalpha(V)beta(3) targeting properties were assessed in nude mice with subcutaneously growing human SK-RC-52 tumors. The multivalent RGD-dendrimers were found to have enhanced affinity toward the alpha(V)beta(3) integrin receptor as compared to the monomeric derivative as determined in an in vitro binding assay. In case of the DOTA-conjugated (111)In-labeled RGD-dendrimers, it was found that the radiolabeled multimeric dendrimers showed specifically enhanced uptake in alpha(V)beta(3) integrin expressing tumors in vivo. These studies showed that the tetrameric RGD-dendrimer had better tumor targeting properties than its dimeric and monomeric congeners