From 2011.igem.org

Redesigning Kumamolisin to Have Higher Activity at Low pH

A Sample Mutation in Foldit Showing a Change from Glycine to Serine

Using Foldit to Design Mutations

In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.

Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.

Mutagenizing Kumamolisin

Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.

Overview of how Kunkel Mutagenesis works

Producing ssDNA

The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. To do this, we designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene by growing the vector harboring our gene in a uracil-N-glycosidase (UNG) and deoxyuracil triphosphate pyrophosphatase (DUT) deficient E. coli- strain. These cells thus did not have the ability to maintain thymine in their DNA, and so all T’s become replaced by U’s in the DNA of these cells. We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.

Kunkel Mutagenesis

Using our isolated ssDNA as a template, we annealed our mutagenic oligos to allow for specific binding of our oligos to our template. The oligo harboring the desired point mutations was then extended, using the wild-type gene and associated vector as a template, by DNA polymerase. This hybrid vector, with one strand of the native Kumamolisin gene and the other a variant containing point mutations, was transformed into UNG and DUT wild-type E. coli, which have the ability to degrade sections of DNA that contain uracil, and replace them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.

Using a Whole Cell Lysate Assay to Test Activity of Mutants

Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data. File:Washington Assay.png

Testing Purified Mutants to Accurately Assess Activity

Purification

After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells. The proteins were then collected on a column, washed with buffer, and eluted off the column. Finally, we dialyzed the protein in a pH 4 buffer.

Assay

Once we had pure protein, we determined the concentrations. We then diluted the proteins to the same concentration. We also used purified wild-type kumamolisin and ScPEP and ran the assay at pH 4. Using the resulting data, we were able to determine which mutants had higher activity than kumamolisin and by how much their activity was greater.