Only the first 1190 amino acids were back translated into nucleotide sequence because this part codes for a soluble protein. Note: the sequence given was a fully optimized sequence and may contains runs of Gs and Cs.
SDS-PAGE and Western Blot
Expression not determined
Strong expression in human cells.
surface spike glycoprotein
To improve expression
The first 1190 amino acids were back translated to nucleotide sequence. The DNA sequence was codon
optimized for mammalian cell expression, replacing the natural codons with the following optimum
codons: ala (gcc), arg (cgc), asn (aac), asp (gac), cys (tgc), glu (gag), gln (cag)m gly (ggc), his
(cac), ile (atc), leu (ctg), lys (aag), met (atg), phe (ttc), pro (ccc), serine (tcc), thr (acc),
trp (tgg), tyr (tac) and val (gtg). When runs of Gc and Cs occurred, suboptimal codons were used.
Babcock, G. J.; Esshaki, D. J.; Thomas, W. D., Jr.; Ambrosino, D. M.
Massachusetts Biologic Laboratories, University of Massachusetts Medical School, Jamaica Plain, Massachusetts 02130, USA. firstname.lastname@example.org
Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor
A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.
J Virol. 78(9): 4552-60.
The soluble part of S glycoprotein was codon optimized for expression in mammalian cells and gain significant yields in human HEK-293T cells.