Preparative parallel protein purification (P4).
In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.
Strömberg
Patrik
P
Global Protein Science and Supply, AstraZeneca R&D Södertälje, SE-15185 Södertälje, Sweden.
Rotticci-Mulder
Joke
J
Björnestedt
Robert
R
Schmidt
Stefan R
SR
D001331:Automation; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002919:Chymotrypsinogen / Q000302:isolation & purification; D003207:Conalbumin / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000302:isolation & purification; D009113:Muramidase / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D014361:Trypsin Inhibitors / Q000302:isolation & purification
D011506:Proteins; D014361:Trypsin Inhibitors; D003207:Conalbumin; D002919:Chymotrypsinogen; D009113:Muramidase; D005634:Fructose-Bisphosphate Aldolase