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Project Design

University of Michigan iGEM Team

Our designed construct consist of a secretion tag, OsmY, at the N-terminus followed by a His-Tag, TEV cleavage site and the desired protein at the C-terminus.

As the construct gets translated, the signal sequence of OsmY will signal the cell to carry the ribosome at the inner membrane. The remaining of the construct will be translated into the periplasm, allowing the protein of interest to fold in an oxidative environment and form disulfide bonds. Further on, the OsmY-tag will carry the construct out of the periplasm into the media through an unknown mechanism.

Once outside the cell, the protein can be harvested simply by centrifuging the growth media to separate the cells from the extracellular proteins. The supernatant is then run through a nickel column, to which the construct will bind due to the His-Tag, therefore separating the desired protein from extracellular contaminants. After eluting the construct from the column, TEV-His protease (a TEV protease linked to a poly-histidine tag) is added to the solution in order to cleave off the desired protein. After cleavage, the solution is run through a nickel column once more; the TEV-His and the construct fragment containing the tags will bind to the column while our desired protein run directly through the column without binding.

In this project, we’ve used an antibody scFv as our protein of interest. To create a single chain scFv we’ve insert the sequence of a heavy variable chain upstream of the sequence of a complementary light variable chain and linked them with a DNA fragment encoding a Myc tag. The Myc tag not only allowed spacing between the 2 antibody fragment but also enable detection of the scFv.