Expression and membrane integration of SARS-CoV M protein

Ma, Hsin-Chieh; Fang, Chiu-Ping; Hsieh, Yi-Ching; Chen, Shih-Chi; Li, Hui-Chun; Lo, Shih-Yen

SARS-CoV M gene fragment was cloned and expressed as a recombinant protein fused with a V5 tag at the C-terminus in Vero E6 cells. In addition to un-glycosylated and glycosylated proteins, one product with smaller size initiated in-frame from the third Met residues probably through ribosomal re-initiation was also detected. Translation initiated in-frame from the third Met is unusual since the sequence around the first Met of SARS-CoV M protein contains the optimal consensus Kozak sequence. The function of this smaller translated product awaits further investigation. Similar to other N-glycosylated proteins, glycosylation of SARS-CoV M protein was occurred co-translationally in the presence of microsomes. The SARS-CoV M protein is predicted as a triple-spanning membrane protein lack of a conventional signal peptide. The second and third trans-membrane regions (a.a. 46–68 and 78–100) are predicted to be the primary type helices, which will be able to penetrate into membrane by themselves, while the first trans-membrane region (a.a. 14–36) is predicted to be the secondary type helix, which is considered to be stabilized by the interaction with other trans-membrane segments. As expected, the second and third trans-membrane regions were able to insert a cytoplasmic protein into the endoplasmic reticulum membrane more efficiently than the first one. These results should be important for the study of SARS-CoV morphogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11373-008-9235-1) contains supplementary material, which is available to authorized users.