University of Reading

1
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.
SDS PAGE
Culture
LB Kanamycin Pre-culture
5x
2L
Kanamycin
Ampicillin
37ºC overnight
Shaking at 37ºC for 3h
LB Kanamycin Single Collony
Pre-culture
Shaking overnight at 37ºC
KDa
200 116 96 66 45 31 21 14 6

• 2ºC - 4ºC
• 4,000 r.p.m.
• 15 minutes

University of Reading

Large amounts of protein