Engineering for Improved Folding of a Human Prolactin Antagonist
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| Language: | English |
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The Ohio State University / OhioLINK
2005
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1392968846 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1392968846 |
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oai_dc |
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NDLTD |
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English |
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NDLTD |
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Biochemistry |
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Biochemistry DePalatis, Laura Engineering for Improved Folding of a Human Prolactin Antagonist |
| author |
DePalatis, Laura |
| author_facet |
DePalatis, Laura |
| author_sort |
DePalatis, Laura |
| title |
Engineering for Improved Folding of a Human Prolactin Antagonist |
| title_short |
Engineering for Improved Folding of a Human Prolactin Antagonist |
| title_full |
Engineering for Improved Folding of a Human Prolactin Antagonist |
| title_fullStr |
Engineering for Improved Folding of a Human Prolactin Antagonist |
| title_full_unstemmed |
Engineering for Improved Folding of a Human Prolactin Antagonist |
| title_sort |
engineering for improved folding of a human prolactin antagonist |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2005 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1392968846 |
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AT depalatislaura engineeringforimprovedfoldingofahumanprolactinantagonist |
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1719435628063490048 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu13929688462021-08-03T06:22:23Z Engineering for Improved Folding of a Human Prolactin Antagonist DePalatis, Laura Biochemistry Human prolactin (hPRL) is a 23 kilodalton protein secreted by the anterior pituitary gland. Its structure is a four alpha helix bundle with an up-up-down-down motif. The molecule contains three disulfide bonds. The main function of hPRL is to aid in the development and maintenance of mammary cells and to initiate lactation. Human prolactin has also been recognized for its involvement in the growth and proliferation of many types of cancers, predominately breast cancer. For this reason, there is much interest in creating prolactin analogs that antagonize the growth of cancer cells.Several prolactin antagonists have been developed in various labs. The prolactin antagonist most important to this document is 41-52 hPRL, in which the amino acid residues 41-52 have been deleted. This molecule has been proposed to work by interfering with one of the receptor binding sites on the prolactin molecule. Preparation of this molecule is difficult because the purified protein contains a large fraction of biologically inactive dimer (usually greater than 50% of the protein expressed is in the dimer form). This is presumably due to strain within the molecule resulting from such a large deleted region. The dimers can be reduced to their monomeric forms using mild reducing conditions on polyacrylamide gel leading to the assumption that the dimeric are held together by an intermolecular disulfide linkage. If the cysteine residues that would normally be involved in an intramolecular disulfide bond are instead free for other types of interactions, they can form disulfide bonds with similar cysteine residues from other prolactin molecules, resulting in a dimeric species.The goal of this project was to engineer the 41-52 hPRL molecule so that it would recover some of the folding capabilities of the wild-type compound that would allow correct disulfide formation while retaining the antagonist biological activity of 41-52 hPRL. To do this, several mutant proteins were created by inserting various amino acids into the 41-52 region. Four initial mutants added a series of glycine residues (2, 3, 4, and 5 glycines were added to the 41-52 hPRL molecule). Another mutation strategy involved adding two alanine residues instead of glycines and yet another mutation added a beta turn (serine-proline-glycine-glycine) to mimic the larger turn created by residues 41-52. Two other mutations involved returning native residues to the deleted region in an attempt to take advantage of native folding.To study the folding of the proteins, the ultraviolet absorbences at 280 nm and 260 nm were observed while purifying the proteins using anion-exchange chromatography. The relative amounts of monomeric and dimeric species were then measured by purifying the protein over a gel filtration column to separate the two species. The identity and relative amount of contamination in the monomer species was studied using SDS-containing gel electrophoresis and the identity of each protein was further elucidated using total protein mass spectrometry. The folding and structure was also assessed using ultraviolet absorbance, fluorescence emission, and circular dichroism spectroscopy. Finally, the biological activity of each of the mutant proteins was assessed using an agonist biological assay with cells expressing the prolactin receptor.While many of the proteins that had increased folding capability resembled wild-type hPRL in the biological assay, the 4G mutant not only proved to be a well-folded molecule but also had a similar biological activity to 41-52 hPRL. This information shows some promise in designing second-generation 41-52 hPRL antagonists for possible therapeutic use. 2005 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1392968846 http://rave.ohiolink.edu/etdc/view?acc_num=osu1392968846 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |