Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System

Razali, Rafida; Subbiah, Vijay Kumar; Budiman, Cahyo

doi:10.3390/data6090099

Cited by 8 publications

(9 citation statements)

References 24 publications

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“…The contaminants in the extract might interfere with the real activity of the enzyme. Purification of this enzyme will almost certainly improve its specific activity, according to Razali et al (2021), who found that purification of recombinant protein increased its specific activity. As for comparisons, the amylase activity of Bacillus sp.…”

Section: Resultsmentioning

confidence: 99%

Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia

et al. 2022

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Abstract. Fazal BZ, Budiman C, Amin Z, Ling CMW. 2022. Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia. Biodiversitas 23: 2807-2815. Thermostable ?-amylases are being used in a wide range of industries, including food, textiles, detergents, pharmaceuticals, and fine chemicals. A good source of thermostable ?-amylases in thermophilic bacteria is found in high-temperature habitats like hot springs. Hence, this study aimed to screen, isolate, and characterize amylase-producing bacteria (APB) from the Poring hot spring in Sabah. Sediment and water samples were collected from the hot springs, serially diluted, plated onto the Luria Bertani agar medium containing starch, and incubated at 60°C for 48 hours. The amylase-producing bacterium was identified by the halo formation around the colony after the agar medium was stained with Lugol's solution. Nine colonies were found to be able to form halo zones, with a creamy colony (A7 strain) producing the highest amylolytic index (4.24). Further characterization of the A7 strain showed that the isolate was a Gram-positive, rod-shaped bacterium, with a positive reaction upon oxidase and catalase tests. The 16S rRNA sequence showed that the A7 strain had 99.81% similarity with the Anoxybacillus flavithermus, and therefore identified as A. flavithermus A7 strain. Further, the growth curve analysis indicated that the A7 strain grew well at 60°C. The 3, 5-dinitrosalicyclic acid (DNS) assay showed the crude enzyme secreted by the A7 strain exhibited optimum amylase activity at 60°C with 8.6 x 10-2 U/ml. This is the first APB obtained from hot springs in Sabah and promising for further studies and applications.

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Section: Resultsmentioning

confidence: 99%

Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia

et al. 2022

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“…Ngay từ khi SARS-CoV-2 được phát hiện, đã có nhiều công trình nghiên cứu biểu hiện 3CLpro tái tổ hợp. Các nghiên cứu cũng cho thấy enzyme chủ yếu được biểu hiện bằng vật chủ E. coli ở dạng dung hợp với 6xHis-tag hoặc glutathione transferase (GST) hoặc maltose-binding protein (MBP) [7][8][9][10][11]. Bên cạnh đó, nhiều đột biến đã xuất hiện trên gen mã hóa 3CLpro của các biến chủng SARS-CoV-2 nên việc thiết lập và cải tiến các quy trình sản xuất 3CLpro vẫn luôn cần thiết [3,12,13].…”

Section: Mở đầU *unclassified

“…rằng 6xHis-3CLpro có mặt chủ yếu trong các pha không hòa tan (Hình 3 A, giếng 4). Tương tự nghiên cứu của chúng tôi, một số nghiên cứu trước đó đã biểu hiện 3CLpro dung hợp với 6xHis-tag tại đầu N ở vi khuẩn E. coli bằng các vector pET21a [7], vector pET-SUMO [8,10] hoặc tại đầu C bằng vector pET32b [9,11]. Trong các nghiên cứu này, 3CLpro tái tổ hợp đều có thể đã được tối ưu hóa codon và biểu hiện ở dạng hòa tan.…”

Section: Nghiên Cứu Biểu Hiện Gen Mã Hóa 3clpro Bằng Pet28aunclassified

“…Trong các nghiên cứu này, 3CLpro tái tổ hợp đều có thể đã được tối ưu hóa codon và biểu hiện ở dạng hòa tan. Mặt khác, bên cạnh dung hợp với 6xHis-tag để thuận lợi cho tinh sạch bằng cột sắc ký ái lực, protein còn được dung hợp với các tag khác như GST, SUMO, MBP hoặc Thioredoxin [8][9][10][11] giúp tăng mức độ hòa tan và biểu hiện của protein đích. Tuy nhiên, quy trình tinh sạch để thu nhận 3CLpro tái tổ hợp thường gồm một số bước và cần thiết phải loại phần tag dung hợp bằng các protease đặc hiệu [8][9][10][11].…”

Section: Nghiên Cứu Biểu Hiện Gen Mã Hóa 3clpro Bằng Pet28aunclassified

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Cloning and Expression of 3 Chymotrypsin-like Protease of SARS-CoV-2 in E. coli using pET28a Vector

Ly¹,

Giang²,

Trang³

et al. 2023

NST

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The 3 chymotrypsin-like protease (3CLpro) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is one of the primary targets for the development of antiviral drug therapies as it plays a critical role in viral replication. In this study, the gene encoding for SARS-CoV-2 3CLpro (918 bp) was amplified from the cDNA of the virus by polymerase chain reaction (PCR) and cloned into the pGEM-T vector. 3CLpro was then inserted into the expression vector pET28a at the end of the 6 histidine residue encoding sequence to form a fusion protein (6xHis-3CLpro). The 6xHis-3Clpro construct was successfully expressed in E. coli. The expression of 3CLpro was highest when E. coli BL21(DE3) RIL harboring pET28a-3CLpro vector was cultured in LB medium at 20 oC, induced by 1.0 mM Isopropyl thiogalactopyranosie (IPTG) when cell density measured by optical density at 600 nm (OD600) reached 0.7-0.8 and harvested after 24 hours of induction. The recombinant 3CLpro was purified by Ni-sepharose affinity chromatography under denaturation conditions. The purified 3CLpro showed to have a 41 kDa band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using polyclonal anti-3CLpro antibody and hydrolyzed a fluorescent specific substrate of 3CLpro after renaturation.

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“…Nevertheless, further experimental confirmation on inhibitory activities of microbial-based compounds against SARS-CoV-2 proteases remains to be conducted. For this purpose, our previous success in the production of recombinant 3CLpro-CoV2 and PLpro-CoV2 [143] is advantageous for further compound testing.…”

Section: Possibility Of Inhibitors Targeting Hiv Protease For Sars-cov2 Proteasesmentioning

confidence: 99%

Structure-Function Characteristics of SARS-CoV-2 Proteases and Their Potential Inhibitors from Microbial Sources

2021

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The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is considered the greatest challenge to the global health community of the century as it continues to expand. This has prompted immediate urgency to discover promising drug targets for the treatment of COVID-19. The SARS-CoV-2 viral proteases, 3-chymotrypsin-like protease (3CLpro) and papain-like cysteine protease (PLpro), have become the promising target to study due to their essential functions in spreading the virus by RNA transcription, translation, protein synthesis, processing and modification, virus replication, and infection of the host. As such, understanding of the structure and function of these two proteases is unavoidable as platforms for the development of inhibitors targeting this protein which further arrest the infection and spread of the virus. While the abundance of reports on the screening of natural compounds such as SARS-CoV-2 proteases inhibitors are available, the microorganisms-based compounds (peptides and non-peptides) remain less studied. Indeed, microorganisms-based compounds are also one of the potent antiviral candidates against COVID-19. Microbes, especially bacteria and fungi, are other resources to produce new drugs as well as nucleosides, nucleotides, and nucleic acids. Thus, we have compiled various reported literature in detail on the structures, functions of the SARS-CoV-2 proteases, and potential inhibitors from microbial sources as assistance to other researchers working with COVID-19. The compounds are also compared to HIV protease inhibitors which suggested the microorganisms-based compounds are advantageous as SARS-CoV2 proteases inhibitors. The information should serve as a platform for further development of COVID-19 drug design strategies.

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Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System

Cited by 8 publications

References 24 publications

Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia

Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia

Cloning and Expression of 3 Chymotrypsin-like Protease of SARS-CoV-2 in E. coli using pET28a Vector

Structure-Function Characteristics of SARS-CoV-2 Proteases and Their Potential Inhibitors from Microbial Sources

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