Increasing Expression Yields of Circularly-Permuted Myoglobins
The permuted myoglobin HGL16 is known to be structurally and functionally similar to the wild-type (wt) sperm whale myoglobin (swMb) yet is less stable to chemical denaturation by 5.2 kcal/mole. Given published reports that stabilities of myoglobin mutants are correlated to expression yields and our...
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| Format: | Text |
| Language: | English |
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Western CEDAR
2003
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| Online Access: | https://cedar.wwu.edu/wwu_honors/248 https://cedar.wwu.edu/cgi/viewcontent.cgi?article=1248&context=wwu_honors |
| Summary: | The permuted myoglobin HGL16 is known to be structurally and functionally similar to the wild-type (wt) sperm whale myoglobin (swMb) yet is less stable to chemical denaturation by 5.2 kcal/mole. Given published reports that stabilities of myoglobin mutants are correlated to expression yields and our own confirmation of this correlation, we are eager to increase expression yields of our destabilized permutants to facilitate protein characterization. One method, fusing HGL16 to Maltose-Binding Protein (MBP), increases yields but appears to destabilize the fused HGL16. Another method for increasing yields, overexpressing protein in the form of inclusion bodies, is particularly useful as it yields apo-myoglobin (apoMb), necessary for investigating true two-state unfolding and the apoMb pH 4.2 intermediate. Urea denaturation studies show our methods of purification are valid, but pH denaturations of our directly expressed, purified apo wt myoglobin remain inconclusive. Further, mutating the distal histidine to phenylalanine (H64F) has been shown to increase the stability of wt swMb, at the cost of impaired function. Introducing an H64F mutation into the HGL16 permutein results in soluble protein with similar expression yields to HGL16 but with a shifted heme absorbance. Preliminary characterization studies yield a reproducible and non- cooperative denaturation curve for holo-H64F-HGL16. |
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