User:Jeff Quinn: Difference between revisions
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*End-to-End Fusion vs. Domain Insertion: | *End-to-End Fusion vs. Domain Insertion: | ||
**end-to-end fusion takes two proteins and fuses them together, often with a linkage, such that the each protein sequence (domain) is intact and separate. this nearly always guarantees that all parts of the chimeric protein will function as their native constituents. Examples of this include fluorescently-tagged proteins or Pericam, which end-to-end fuses calmodulin, M13, and a circularly-permuted EYFP. | **end-to-end fusion takes two proteins and fuses them together, often with a linkage, such that the each protein sequence (domain) is intact and separate. this nearly always guarantees that all parts of the chimeric protein will function as their native constituents. Examples of this include fluorescently-tagged proteins or Pericam, which end-to-end fuses calmodulin, M13, and a circularly-permuted EYFP. | ||
**Domain insertion however | **Domain insertion, however, involves taking the sequence from one protein (insert domain) and inserting it into the middle of another (host? better word?) protein, such that the host protein's conformation and functionality are greatly disrupted. The advantage of this is that this new chimeric protein can adopt new behavior that reflects both the insert protein's and the host protein's behaviors, but only when the respective domains "cooperate" with eachother. For example, Guntas and Ostermeier took an enzyme that hydrolyzes penicillin-like antibiotics and inserted a protein that binds to maltose, a sugar molecule. After a library* of chimera proteins was produced, two such proteins showed new behavior: the antibiotic hydrolyzing enzyme would only break down antibiotics if the maltose-binding protein was bound to maltose. In effect, this creates a molecular switch such that the enzymatic activity can be controlled (turned off or on) by maltose. | ||
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===Research Problem and Goals=== | ===Research Problem and Goals=== | ||
Revision as of 14:39, 28 April 2008
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Contact Info
Jeffrey Quinn, MIT 2010, Course 20
- (916)765-9607
- jquinn[at]mit.edu
Module 3 Research Proposition Information
Project Overview
- By inserting the sequence of one protein into the sequence of another, the functionality of the protein can be lost; however, the new chimeric protein can assume new characteristics. For example, by inserting the sequence for an allosteric substrate-binding protein into the sequence of a metabolic enzyme, it is possible that the two proteins together can behave in a new way: as a switch. When substrate binds to the chimera, it can induce a conformational change in the enzyme that either activates or inactivates its enzymatic behavior, effectively creating a novel control mechanism for modulating the enzyme's activity with an input molecule (the substrate).
- Based on the experiment described by Guntas and Ostermeier, we plan to develop our own "molecular switch" protein in the same way that they did, by creating an allosteric enzyme that "couples effector levels (input) to enzyme activity (output)."
- Enzymes to work with (IDEAS):
- hemoglobin
- EGFP (easy to moniter!)
- pyruvate decarboxylase (alcohol fermentation?)
- insulin
- Substrate-Binding proteins to work with (IDEAS):
- calmodulin
- oxygen sensor?
Background Information
- End-to-End Fusion vs. Domain Insertion:
- end-to-end fusion takes two proteins and fuses them together, often with a linkage, such that the each protein sequence (domain) is intact and separate. this nearly always guarantees that all parts of the chimeric protein will function as their native constituents. Examples of this include fluorescently-tagged proteins or Pericam, which end-to-end fuses calmodulin, M13, and a circularly-permuted EYFP.
- Domain insertion, however, involves taking the sequence from one protein (insert domain) and inserting it into the middle of another (host? better word?) protein, such that the host protein's conformation and functionality are greatly disrupted. The advantage of this is that this new chimeric protein can adopt new behavior that reflects both the insert protein's and the host protein's behaviors, but only when the respective domains "cooperate" with eachother. For example, Guntas and Ostermeier took an enzyme that hydrolyzes penicillin-like antibiotics and inserted a protein that binds to maltose, a sugar molecule. After a library* of chimera proteins was produced, two such proteins showed new behavior: the antibiotic hydrolyzing enzyme would only break down antibiotics if the maltose-binding protein was bound to maltose. In effect, this creates a molecular switch such that the enzymatic activity can be controlled (turned off or on) by maltose.
