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2 protocols using benchtop cooling centrifuge

1

Purification and Characterization of MERS-CoV PL-pro

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The ORF of MERS-CoV PLpro (1484–1800 polyprotein residues, GenBank accession number NC_019843.2) was cloned into pET28a plasmid under T7 promoter as published before (Lin et al. 2014 (link)). The codon was optimized (GenScript, USA) and cloned between NcoI and XhoI sites which was in frame of C-terminal His tag present on the vector. E. coli BL21 (DE3) pLysS was used for the expression of recombinant protein. Low-molecular weight protein markers, prepacked Ni-NTA, and Superdex 75 columns were from Amersham Biosciences (United Kingdom). Chicken egg lysozyme was from USB Corporation, USA. Benzonase, ANS, and kanamycin from Sigma. IPTG was purchased from Bio Basic, Canada. All other chemicals used in this study were of reagent grade. Cary 60 spectrometer and Cary Eclipse spectrofluorometer were from Agilent technologies, USA. AKTA purification system was from Amersham Biosciences (United Kingdom) and SDS-PAGE assembly from Bio-Rad (USA). Thermomixer and benchtop cooling centrifuge were from Eppendorf, Germany. Innova 44R Shaking incubator was from New Brunswick, Germany. Chirascan-Plus spectropolarimeter was from Applied photophysics, United Kingdom.
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2

Expression and Purification of cHSPA6

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The ORF of cHSPA6 cloned on pET15 vector and expression host E. coli BL21 (DE3) pLysS were kindly provided by Elrobh et al. (2011) (link). IPTG and ampicillin were obtained from Biobasic. Benzonase was purchased from Sigma, Chicken egg lysozyme from USB Corporation. Superdex 75, Ni–NTA resin, low molecular weight markers and prepacked columns were from Amersham Biosciences. All other chemicals used in this study were of reagent grade. Ultrospec 2100 pro Spectrophotometer, AKTA purification system, SDS–PAGE assembly were from Amersham Biosciences. Thermomixer, electroporator and benchtop cooling centrifuge were from Eppendorf. Lamp sterilizer from Cole-Parmer, shaking incubator from Jeio Tech, South Korea, gel scanner from Epson and pH meter was from Sentron.
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