Team:Washington/Celiacs/Methods

From 2011.igem.org

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(Kunkel Mutagenesis)
(Redesigning Kumamolisin to Have Higher Activity at Low pH)
 
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=Redesigning Kumamolisin to Have Higher Activity at Low pH=
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<center><big><big><big><big>'''Gluten Destruction: Methods'''</big></big></big></big></center><br><br>
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[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]
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==Using Foldit to Design Mutations==
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='''Redesigning Kumamolisin to Have Higher Activity at Low pH'''=
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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.  
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[[File:Washington Foldit.png|550px|thumb|right|A Sample Mutation in [http://fold.it Foldit] Showing a Change from Glycine to Serine]]
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=='''Using [http://fold.it 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 [http://fold.it 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.
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.
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Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.
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==Mutagenizing Kumamolisin==
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----
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='''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.
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.
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[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]
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[[File:Washington Kunkels.png|500px|thumb|left|Overview of how Kunkel Mutagenesis works]]
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===Producing ssDNA===
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=='''Kunkel Mutagenesis'''==
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.  
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.  
-
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:
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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.  
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*isolated the single stranded DNA (ssDNA) of the sense strand of our gene,
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*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, and finally,
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To incorporate these mutations, we first isolated single stranded DNA (ssDNA) of our vector harboring the wild-type Kumamolisin gene. To do this we infected cells with bacteriophage M13, which packages its own ssDNA genome identified by length, and so in tandem packaged our vector in single stranded form. We then harvested the phage from the lysed culture of E. coli, and extracted our single stranded vector DNA.
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*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.
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Next, we annealed and extended our mutagenic oligos to incorporate the specified mutations into the newly synthesized antisense strand. This hybrid vector was transformed into E. coli that degraded the original uracil-containing DNA and replaced it with sections complementary to the mutagenized strand.
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----
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='''Using a Whole Cell Lysate Assay to Test Activity of Mutants'''=
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To test our designs, we developed a whole cell lysate assay that allowed us to perform a rough screen of a large number of mutants. In this assay, we expressed our mutant enzymes in <i>E. coli</i>, lysed the cells and separated the enzymes from large cell particulate. We then performed the assay at pH 4, mimicking the gastric environment. We added our model PQLP peptide, conjugated to both a fluorophore and a quencher so that no fluorescence would be achieved until after the peptide had been enzymatically cleaved. We then measured the fluorescence of each reaction at 30 second intervals, and were thereby able to estimate relative activity on breaking down PQLP by increase in fluorescence of the system.
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[[File:Washington Whole Cell Lysate Assay.jpg|center|General Overview of the Whole Cell Lysate]]
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----
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===Kunkel Mutagenesis===
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='''Testing Purified Mutants to Accurately Assess Activity'''=
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We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced 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.
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[[File:Washington First Raw Data.png|right|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]
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==Using a Whole Cell Lysate Assay to Test Activity of Mutants==
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=='''Purification'''==
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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.
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From our whole cell lysate screen of each design, we identified mutants that showed the most increase in activity from the wild-type Kumamolisin. We then proceeded to purify these most promising variants and test them against the wild-type and against SC PEP using the same fluorescence metric designed for the whole cell lysate assay. The key difference between the whole cell assay and the purified protein assay is that in the latter we were able to control the concentration of enzyme in each well, adjusting for the possibility of varying expression levels and thus enzyme concentrations in the whole cell lysate assay.
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[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]
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==Testing Purified Mutants to Accurately Assess Activity==
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Purification was performed via Nickel-affinity chromatography, and resulting protein concentrations were measured using ultraviolet-visible spectrophotometry.
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===Purification===
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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, followed by collection of the purified proteins.
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===Assay===
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=='''Assay'''==
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Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.
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Concentration dependent assays were performed for each promising mutant. We measured the fluorescence of each reaction at 30 second intervals to see the rate at which fluorescence increased, thus obtaining a relative rate of cleavage of PQLP by increase in fluorescence of the system. Raw data appeared as shown right, and the slope of each line was calculated, giving us relative rate information that could be used in conjunction with rate information obtained in the same assay for native Kumamolisin to determine fold change in activity.

Latest revision as of 02:36, 29 September 2011


Gluten Destruction: Methods


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




Kunkel Mutagenesis

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.

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.

To incorporate these mutations, we first isolated single stranded DNA (ssDNA) of our vector harboring the wild-type Kumamolisin gene. To do this we infected cells with bacteriophage M13, which packages its own ssDNA genome identified by length, and so in tandem packaged our vector in single stranded form. We then harvested the phage from the lysed culture of E. coli, and extracted our single stranded vector DNA.

Next, we annealed and extended our mutagenic oligos to incorporate the specified mutations into the newly synthesized antisense strand. This hybrid vector was transformed into E. coli that degraded the original uracil-containing DNA and replaced it with sections complementary to the mutagenized strand.








Using a Whole Cell Lysate Assay to Test Activity of Mutants

To test our designs, we developed a whole cell lysate assay that allowed us to perform a rough screen of a large number of mutants. In this assay, we expressed our mutant enzymes in E. coli, lysed the cells and separated the enzymes from large cell particulate. We then performed the assay at pH 4, mimicking the gastric environment. We added our model PQLP peptide, conjugated to both a fluorophore and a quencher so that no fluorescence would be achieved until after the peptide had been enzymatically cleaved. We then measured the fluorescence of each reaction at 30 second intervals, and were thereby able to estimate relative activity on breaking down PQLP by increase in fluorescence of the system.

General Overview of the Whole Cell Lysate




Testing Purified Mutants to Accurately Assess Activity

We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.

Purification

From our whole cell lysate screen of each design, we identified mutants that showed the most increase in activity from the wild-type Kumamolisin. We then proceeded to purify these most promising variants and test them against the wild-type and against SC PEP using the same fluorescence metric designed for the whole cell lysate assay. The key difference between the whole cell assay and the purified protein assay is that in the latter we were able to control the concentration of enzyme in each well, adjusting for the possibility of varying expression levels and thus enzyme concentrations in the whole cell lysate assay.

Purification was performed via Nickel-affinity chromatography, and resulting protein concentrations were measured using ultraviolet-visible spectrophotometry.

Assay

Concentration dependent assays were performed for each promising mutant. We measured the fluorescence of each reaction at 30 second intervals to see the rate at which fluorescence increased, thus obtaining a relative rate of cleavage of PQLP by increase in fluorescence of the system. Raw data appeared as shown right, and the slope of each line was calculated, giving us relative rate information that could be used in conjunction with rate information obtained in the same assay for native Kumamolisin to determine fold change in activity.