1. Large amounts of protein
Structural Studies of Agricultural Target Proteins
M.I.Moraes,C. J. Cardin, Andrew Pannifer
School of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, U.K.
Crystallography
Cloning
Purification
Re-suspend
Cells (15.7g)
Batch purification
High pressure
cell disruptor
SPIN
Gel Filtration Chromatography
Conductivity/Temp/PH
Spect. UV
Pressure
Expression
X- ray crystallography plays a crucial role in modern molecular biology. This method became vital for studying the three-dimensional structure of macromolecules like proteins. However, to obtain good quality crystals we must obtain proteins with good purity levels.
Cloning, expression and purification of recombinant proteins facilitates production. Consequently a wide variety of cloning methods and expression systems have been developed as well as purification methods.
The aim of this project is to perform structural studies of agricultural proteins applying the methods and techniques mentioned above.
Culture
SDS – PAGE LB +
Kanamycin
Pre-culture
Kanamycin
Ampicillin 5x 2L
37ºC overnight
Shaking at 37ºC for 3h LB +
Kanamycin
Single Collony
Pre-culture
Shaking overnight at 37ºC
KDa
200 45 31 21 14 6
2ºC - 4ºC
4,000 r.p.m.
15 minutes
University of Reading =
Large amounts of protein