The expression and purification of HIV gp41 protein (Ala517-Asn711, GenBank accession number P03377.1) were performed as described previously (38). Briefly, the gene encoding gp41 protein was synthesized by BGI (Beijing, China) and cloned into the pET21a (+) vector. N-terminal methionine was added, and Cys603 and Cys609 were mutated to Ala. BL21 (DE3) was used as the host strain for gp41 expression. Expressed protein was extracted with SDS and purified by a combination of gel filtration and hydroxyapatite chromatographies. The protein was folded by exchanging SDS for Fos-Choline-12 (DPC; Affymetrix) and concentrated to 10 mg/ml with an Amicon-Ultra 15 ml, 30-kDa concentrator (EMD Millipore). The EBOV GP (catalog no. 40304-V08B1) and influenza virus HA (catalog no. 11055-V08B) used for the photoaffinity labeling were purchased from Sino Biological Inc. As shown in the product specifications, the baculovirus-insect cell expression system was used to express the EBOV GP (Met1-Gln650, GenBank accession number AAC54887.1) and influenza virus HA (Met1-Gln529, GenBank accession number ACP41105.1). The secondary structures of HIV-1 gp41, EBOV GP, and influenza virus HA were determined by circular dichroism spectroscopy (38). All the three proteins exhibited similar α helicity and β strand to those in the UniProt database, confirming that the proteins have well-fold structures.

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