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J. F. Koretz, Y. Li; Analysis of Modifications of the Alpha-Core Domain Region: Preliminary Studies of Solubility. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2040. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The small heat shock protein superfamily can be divided into two groups, one largely animal and the other largely plant and bacterial, based on the length and sequence of an alpha-core domain involved in beta-strand sharing in the latter group (Salerno et al., 2003; Eifert et al., 2005). We have hypothesized that this region is not essential for assembly or functionality in the animal group, but important in the other. The role of this region in assembly and functionality is therefore being investigated by exchanging it between human alphaA-crystallin and MjHsp16.5.
Multiple alignments of sHsp sequences from all kingdoms were used to delineate the beginning and end of the relevant sequence in the core domain of alphaA-crystallin and MjHsp16.5. Standard molecular biological methods were then used to create a set of chimera for each parent. I.e., for alphaA, the parent sequence is represented as AAA, the swapped sequence as AHA, and the negative control as A-A, while for MjHsp16.5, the equivalent sequences are HHH, HAH, and H-H respectively. Each chimera was prepared in two forms, with a His tag at either the N- or C-terminus to assess its potential effect.
After sequencing to ensure the accuracy of the modifications, the twelve sequences were successfully overexpressed in an E.coli expression system. All the alphaA-crystallin-based chimera (AHA and A-A with either C- or N-terminal His tags) were soluble in the high-salt solutions necessary for Bind purification, while the MjHsp16.5-based chimera (HAH and H-H with either C- or N-terminal His tags) were only marginally soluble.
We have previously demonstrated the robust nature of the alphaA sequence relative to Hsp16.5 in a series of chimera with swapped or absent N-terminal sequences up to the beginning of the consensus region. Our preliminary results suggest that any variation of the beta-strand sharing region being investigated here may be irrelevant to alphaA subunit folding and association, while being critical for MjHsp16.5 and related group members.
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