Mutagenesis study to ameliorate the bacterial expression of phosphoprotein P of vesicular stomatitis virus |
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Authors: | Susanta Pahari D J Chattopadhyay |
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Institution: | (1) Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular road, 700 019 Calcutta, India |
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Abstract: | The phosphoprotein gene of vesicular stomatitis virus, a Rhabdovirus, has been inserted into bacterial expression plasmids
containing the Escherichia coli tac promoter and ribosome binding site (RBS). A low level of expression of the protein was
detected. Sequence analysis showed the presence of 15 nucleotides in the spacer region i.e., between the Shine-Dalgarno sequence
and ATG. Alteration of the distance and the sequence in the spacer region by oligonucleotide-directed mutagenesis revealed
a correlation among the expression levels, accessibility of the RBS and requirement for a minimum spacing of at least 7 nucleotides
between the Shine-Dalgarno sequence and ATG for optimal gene expression. |
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Keywords: | VSVP protein overexpression parameter optimization 5′ untranslated region mutagenesis |
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