(371a) Application of Split Intein in an ELP-Intein System

Changhua Shi, Qing Meng, David Wood

Inteins are internal protein sequences that can auto-excise from precursor proteins and splice together the flanking sequences.  By substitution of amino acids, inteins can be transformed into self-cleavage elements, where cleavage is controlled by pH or thio-reagents.  In our previous work, we have successfully combined a pH sensitive cleaving intein with a non-chromatographic purification tag (ELP, elastin like protein) to create a self-cleaving aggregation tag.  The target protein can be easily purified by several steps of centrifugation and without expensive chromatography steps.  However, the engineered intein usually has some level of pre-cleavage in vivo, depending on the target protein and expression temperature.  This affects the final yield of the protein and the cost of production, especially for some highly expensive pharmaceutical proteins.  This is also a             problem that hampers the use of this technology at large scale.  Here we introduce a split intein into our ELP-intein system to tackle this problem.  The N terminal fragment of intein is removed from the ELP-intein-target protein precursor, and fused to a second ELP tag to be used as a switch for the cleavage activity of the tagged target precursor.  The activity of truncated intein is tightly controlled by addition of the complementary N terminal fragment.  The trans-cleaving efficiency of our split intein is similar to the full-sized intein.  Our study also partially showed that the recognition and association of the intein fragments is strong even though there is a large tag, ELP, between them.  Further, mutation of the first of amino acid of the target protein into the native conserved residues in this site, such as serine and cysteine can increase the cleavage efficiency.  This dual ELP-intein technology can also be applied to do cost-effective protein purification in many other systems, such as yeast and mammalian cells.