Protocol detail

MERS-CoV HR1-L6-HR2 Fusion Protein Expression

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To express the fusion protein HR1-L6-HR2, the nucleotide sequence encoding the MERS-CoV HR2 region (residues 1,245–1,289) was attached to the 3′ end of the HR1 region (residues 984–1,062) with a short linker (L6: SGGRGG) in between by overlapping PCR as previously described for constructing SARS-CoV (or HIV-1) 6-HB (ref. 25 (link)). The whole sequence was subcloned into the pET-28a vector with an artificial SUMO-tag. The pET-28a-SUMO-HR1-L6-HR2-transformed E. coli cells were induced with 1 mM IPTG, incubated overnight at 16 °C and purified by Ni-NTA affinity resin (Qiagen, Valencia, CA, USA). His-SUMO-tag was cleaved off with Ulp1 enzyme at 4 °C for 2 h. The purified HR1-L6-HR2 was applied onto a Superdex-75 gel filtration column (GE Healthcare, Piscataway, NJ, USA). Fractions containing HR1-L6-HR2 trimer were collected and concentrated by ultrafiltration⁵³.

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Lu L., Liu Q., Zhu Y., Chan K.H., Qin L., Li Y., Wang Q., Chan J.F., Du L., Yu F., Ma C., Ye S., Yuen K.Y., Zhang R, & Jiang S. (2014). Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nature Communications, 5, 3067.

Publication 2014

Ni-NTA affinity resin Superdex-75 gel filtration column

Cells E coli Enzyme Gel filtration Hiv 1 Iptg Mers cov Nucleotide sequence Protein Resin Sars cov Vector

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Protocol cited in 2 other protocols

Variable analysis

independent variables

The whole sequence (HR1-L6-HR2) was subcloned into the pET-28a vector with an artificial SUMO-tag.
The pET-28a-SUMO-HR1-L6-HR2-transformed E. coli cells were induced with 1 mM IPTG.
The His-SUMO-tag was cleaved off with Ulp1 enzyme at 4 °C for 2 h.

dependent variables

The purified HR1-L6-HR2 was applied onto a Superdex-75 gel filtration column.
Fractions containing HR1-L6-HR2 trimer were collected and concentrated by ultrafiltration.

control variables

The pET-28a-SUMO-HR1-L6-HR2-transformed E. coli cells were incubated overnight at 16 °C.
The purification was performed using Ni-NTA affinity resin (Qiagen, Valencia, CA, USA).

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