Dataset Information

Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.

ABSTRACT: The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 ?M with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoid compounds were 47-381 ?M. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47 ?M, respectively. GCG showed a competitive inhibition pattern with K ( i ) value of 25 ± 1.7 ?M. In molecular docking experiments, GCG displayed a binding energy of -14 kcal mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.

SUBMITTER: Nguyen TT

PROVIDER: S-EPMC7087583 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.

Nguyen Thi Thanh Hanh TT Woo Hye-Jin HJ Kang Hee-Kyoung HK Nguyen Van Dao VD Kim Young-Min YM Kim Do-Won DW Ahn Sul-Ah SA Xia Yongmei Y Kim Doman D

Biotechnology letters 20120215 5

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 μM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds ...[more]

PMID: 22350287

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Project description:BackgroundPichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris.ResultsA non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (PAOX1UP-g2, PTEF1UP-g1, and PFLD1UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR-Cas9-mediated gene co-integration method in P. pastoris.ConclusionsA CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

Dataset Information

Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.

Publications

Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.

Similar Datasets

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