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Purification of Violaxanthin De-epoxidase expressed in E.Coli and identification of disulfide bonds using MS

Hallin, Erik LU (2009) KEMX03 20091
Department of Chemistry
Abstract
Plants need light to convert earbon dioxide to organic compounds, but when exposed to too much light the photosynthetic machinery takes damage. This is prevented by conversion of violaxanthin to zeaxanthin, which participate in a process that converts the excess light into heat. The conversion of violaxanthin to zeaxanthin is done by the enzyme violaxanthin de-epoxidase.

VDE from spinach have been sequenced and expressed in E. coli. After harvesting of periplasmic proteins VDE has been purified using DEAE column, two-step precipitation and lipid affinity precipitation with MGDG.

By not reducing VDE while running it on a SDS-PAGE gel and avoiding reducing substances in purification methods and sample preparation for mass spectroscopy... (More)
Plants need light to convert earbon dioxide to organic compounds, but when exposed to too much light the photosynthetic machinery takes damage. This is prevented by conversion of violaxanthin to zeaxanthin, which participate in a process that converts the excess light into heat. The conversion of violaxanthin to zeaxanthin is done by the enzyme violaxanthin de-epoxidase.

VDE from spinach have been sequenced and expressed in E. coli. After harvesting of periplasmic proteins VDE has been purified using DEAE column, two-step precipitation and lipid affinity precipitation with MGDG.

By not reducing VDE while running it on a SDS-PAGE gel and avoiding reducing substances in purification methods and sample preparation for mass spectroscopy the disulfide bonds should be intact. When analyzing non-reduced VDE with MS new mass peaks should appear, representing masses of two peptides linked tagether with a disulfide bond.

These peaks were found in the spectrum using Peptidemap. Then these peaks were further analyzed with MS/MS and data interpreted with xQuest. Four disulfide bonds could be confirmed with MS/MS according to xQuest. (Less)
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author
Hallin, Erik LU
supervisor
organization
course
KEMX03 20091
year
type
M2 - Bachelor Degree
subject
keywords
Biokemi
language
English
id
3053576
date added to LUP
2012-09-19 11:33:39
date last changed
2012-09-19 11:33:39
@misc{3053576,
  abstract     = {Plants need light to convert earbon dioxide to organic compounds, but when exposed to too much light the photosynthetic machinery takes damage. This is prevented by conversion of violaxanthin to zeaxanthin, which participate in a process that converts the excess light into heat. The conversion of violaxanthin to zeaxanthin is done by the enzyme violaxanthin de-epoxidase.

VDE from spinach have been sequenced and expressed in E. coli. After harvesting of periplasmic proteins VDE has been purified using DEAE column, two-step precipitation and lipid affinity precipitation with MGDG.

By not reducing VDE while running it on a SDS-PAGE gel and avoiding reducing substances in purification methods and sample preparation for mass spectroscopy the disulfide bonds should be intact. When analyzing non-reduced VDE with MS new mass peaks should appear, representing masses of two peptides linked tagether with a disulfide bond.

These peaks were found in the spectrum using Peptidemap. Then these peaks were further analyzed with MS/MS and data interpreted with xQuest. Four disulfide bonds could be confirmed with MS/MS according to xQuest.},
  author       = {Hallin, Erik},
  keyword      = {Biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Purification of Violaxanthin De-epoxidase expressed in E.Coli and identification of disulfide bonds using MS},
  year         = {2009},
}