Discovery and Evaluation of Peptide Ligands for Selective Adsorption and Release of Cas9 Nuclease on Solid Substrates
| Autor: | Sahand Saberi Bosari, Raphael Prodromou, Connor Market, R Ashton Lavoie, Adriana San Miguel, Stefano Menegatti, Mohammad A. Omary, Kevin Day |
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| Rok vydání: | 2019 |
| Předmět: |
Lysis
Streptococcus pyogenes Biomedical Engineering Pharmaceutical Science Bioengineering 02 engineering and technology Ligands medicine.disease_cause Biochemistry 01 natural sciences Substrate Specificity Selective adsorption Nuclease Peptide Library CRISPR-Associated Protein 9 Drug Discovery Escherichia coli medicine Amino Acid Sequence Binding site Cas9 Peptide library Peptide sequence Pharmacology Binding Sites biology 010405 organic chemistry Drug discovery Chemistry Organic Chemistry 021001 nanoscience & nanotechnology Combinatorial chemistry 3. Good health 0104 chemical sciences Molecular Docking Simulation Resins Synthetic CRISPR biology.protein Adsorption Peptides 0210 nano-technology Biotechnology |
| Zdroj: | Datacite Microsoft Academic Graph |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/acs.bioconjchem.9b00703 |
| Popis: | The rapid expansion of CRISPR in biotechnology, medicine, and bioprocessing poses an urgent need for advanced manufacturing of Cas nucleases. The lack of Cas-targeting ligands, however, prevents the development of platform processes for purifying this class of molecules. This work represents the first effort at developing short synthetic Cas9-binding peptides and demonstrates their applicability as affinity ligands for the purification of a Cas nuclease. Candidate Cas9-targeting peptides were initially identified by screening a solid-phase peptide library against a model mixture of Streptococcus pyogenes Cas9 spiked in Escherichia coli cell lysate. An ensemble of homologous sequences was identified, conjugated on Toyopearl resin, and evaluated by Cas9 binding studies to identify sequences providing selective Cas9 capture and efficient release. In silico docking studies were also performed to evaluate the binding energy and site of the various peptides on Cas9. Notably, sequences GYYRYSEY and YYHRHGLQ were shown to target the RecII domain of Cas9, which is not involved in nuclease activity and was targeted as an ideal binding site. The peptide ligands were validated by purifying Cas9 from the E. coli lysate in dynamic conditions and through measurements of binding capacity and strength (Qmax and KD). The resulting values of Qmax = 4-5 mg Cas9 per mL of resin and KD ∼ 0.1-0.3 μM, product recovery (86-89%), and purity (91%-93%) indicate that both peptides, and YYHRHGLQ in particular, can serve as capture ligands in a platform purification process of Cas9. |
| Databáze: | OpenAIRE |
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