Large amounts of protein

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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 200 116 96 66 45 31 21 14 6 2ºC - 4ºC 4,000 r.p.m. 15 minutes University of Reading = Large amounts of protein