RESUMO
Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.
Assuntos
Enzima de Conversão de Angiotensina 2/isolamento & purificação , Dipeptidil Peptidase 4/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Clonagem Molecular , Dipeptidil Peptidase 4/biossíntese , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Plasmídeos/química , Plasmídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Ressonância de Plasmônio de SuperfícieRESUMO
The highly oxygenated pimarane diterpenoids basimarols A, B, and C (3-5) were isolated from the plant species Basilicum polystachyon, which was collected within the Australian arid zone. Structure elucidation was performed using a suite of spectroscopic techniques, including X-ray crystallography. Anticancer and anti-DENV activity of 3-5 was explored, but only limited activity was observed. More extensive antiviral evaluation of stachyonic acid A (1), which was also isolated from B. polystachyon, revealed broad spectrum antiviral activity against West Nile virus (Kunjin strain, WNVKun) and human influenza viruses H1N1 and H3N2.