From 2011.igem.org

Redesigning Kumamolisin to Have Higer Activity at Low pH

Using Foldit to Produce Mutations

In order to create mutations from wild-type kumamolisin, we first loaded the enzyme structure into the protein editing game Foldit. Foldit allows the user to modify the way in which a protein folds by changing the amino acid sequence. Using Foldit, we modified the amino acid residues around the active site of kumamolisin which contained within it a Proline-Glutamine-Lysine-Proline (PQLP) motif found throughout the gliadin peptide of gluten. With the PQLP locked in place along with the key active site amino acid residues, mutations were made which appeared to stabilize PQLP within the active site. Using this method, we produced several mutants at a time which could potentially increase the enzyme's proteolytic activity towards gliadin.

Mutagenizing Kumamolisin

Producing ssDNA

Once we had obtained feasible mutants from the Foldit program, we ordered these mutations in oligonucleotide primers to be used in Kunkel mutagenesis. To perform the mutagenesis, we first transformed the plaasmid containing wild-type kumamolisin into chemically competent cells of Escherichia coli CJ236. These cells were then grown and harvested for their single stranded DNA (ssDNA).

Kunkel Mutagenesis

Using ssDNA as a template, we annealed oligo nucleotides in a PCR block to increase specific binding. The primer was then extended on the ssDNA using T7 DNA polymerase. We then dialyzed the DNA to remove salts introduced in previous steps. We then added the DNA to cuvettes containing electrically competent E. coli BL21* cells. These cells were then pulsed with electricity to allow the uptake of the newly mutagenized DNA. After growing the cells for an hour in TB, we plated the cells on LB agar containing kanamycin.

Using a Whole Cell Lysate Assay to Test Feasability of Mutants

Purifying Mutants to Accurately Assess Activity

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