Secretion of Semliki Forest virus membrane glycoprotein E1 from Bacillus subtilis.
The gene coding for the Semliki Forest virus (SFV) membrane protein E1 was joined to a secretion vector containing the promoter and signal sequence regions of the alpha-amylase gene from Bacillus amyloliquefaciens. To facilitate secretion, the regions coding for the N-terminal signal peptide (the 6K protein) and the C-terminal hydrophobic transmembrane domain of the E1 gene were deleted. After transformation into B. Subtilis, E1 was shown by immunoblotting to be expressed at a low level (about 0.5-1 mg/1). Contrary to what was expected, most of the E1 remained cell-associated. Deletion of a residual 7 C-terminal amino acids from the 6K region neither increased the level of expression nor significantly improved the secretion. Immunofluorescence microscopy of protoplasts prepared from B. subtilis cells expressing E1 suggested that the cell-associated E1 was located at the outer surface of the bacterial membrane. Addition of protease inhibitors to the culture medium somewhat increased the amount of extracellular E1, suggesting that proteolytic degradation of the foreign gene product may be one reason for the low level of expression. This conclusion was also supported by experiments carried out in Bacillus minicells, which indicated that the expression of the E1 gene in the absence of synthesis of bacterial proteases was about the same as that of alpha-amylase expressed from the cloned gene using the same promoter and signal sequence.
D001412:Bacillus subtilis / Q000235:genetics*; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012672:Semliki forest virus / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014765:Viral Vaccines / Q000276:immunology
D006023:Glycoproteins; D011480:Protease Inhibitors; D014764:Viral Proteins; D014765:Viral Vaccines