Presentation Title
Purification of Viral Fibroblast Growth Factors Tagged with Maltose Binding Protein using Cationic Starches Derived from Maize
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Abstract
Tagging proteins of interest with maltose binding protein (MBP) is a common method to produce recombinant proteins in E. coli that can be purified using amylose resin in affinity chromatography with a high degree of purity. The rising price of amylose resin over the years has made this purification method less desirable. Successful methods for purifying MBP-tagged proteins have previously been demonstrated using Catamyl-VS cationic starch derived from tapioca. While this method seems to offer an affordable route for protein purification, finding a cationic starch derived from tapioca starch that meets the specifications of Catamyl-VS has proven itself impossible due to its low demand in the paper making industry here in the United States. This study aims to find a cationic starch derived from corn that is cost effective, readily available in the market, and successful in purifying MBP tagged proteins with a high degree of purity.
Four cationic starches derived from corn (Charge +99, Charge +110, Charge +309, Charge +310) were kindly gifted to us by Cargill. Prior to this study, viral fibroblast growth factor (vFGF) genes from Autographa californica M nucleopolyhedrovirus (AcMNPV) and Choristoneura fumiferana M nucleopolyhedrovirus (CfMNPV) were cloned with a coding sequence for MBP to produce plasmids coding for the fusion proteins Ac6 and Cf6, respectively. Ac6 and Cf6 plasmids were transformed into E. coli ER2508 to express the recombinant proteins. Lysate from Ac6 and Cf6 were incubated with each cationic starch at concentrations of 0.2%, 0.3%, and 0.4% (w/v) cationic starch. Precipitation was done with 10% (w/v) PEG 3350 and 50mM CaCl2. The bound MBP tagged protein was eluted off the cationic starch polymer by resuspension in 1M NaCl and separated by centrifugation. Current Bradford results show protein concentrations as high as 232µg/mL for Cf6 lysate with 0.4% (w/v) +309 cationic starch, and 311µg/mL for Cf6 lysate with 0.3% (w/v) +310 cationic starch. The Cf6 lysate with +99 and +110 cationic starches did not fall within the linear range of the BSA standard. A duplicate purification using Ac6 lysate and only +310 and +310 revealed high protein concentration in a Bradford assay, but high protein concentrations in the “No Starch” control as well. SDS-PAGE Coomassie Staining of both purification tests revealed no purification of the MBP-tagged vFGF protein and high protein precipitation in the no starch control. A lower percent PEG (6%) and lower protein concentrations were determined to run the experiment one more time to determine if we can eliminate the precipitation of unwanted proteins and purify the MBP-tagged vFGF protein. This purification is currently underway and will be followed by protein analysis, SDS-PAGE Coomassie staining, and Western Blotting. If successful, a comparison of the cationic starch purification will be compared to purification by amylose affinity chromatography. If the purification is high, I plan to continue researching the mitogenic effect vFGF has on insect and mammalian cells lines.
College
College of Science & Engineering
Department
Biology
Presentation Type
Video (Prerecorded-MP4)
Purification of Viral Fibroblast Growth Factors Tagged with Maltose Binding Protein using Cationic Starches Derived from Maize
Tagging proteins of interest with maltose binding protein (MBP) is a common method to produce recombinant proteins in E. coli that can be purified using amylose resin in affinity chromatography with a high degree of purity. The rising price of amylose resin over the years has made this purification method less desirable. Successful methods for purifying MBP-tagged proteins have previously been demonstrated using Catamyl-VS cationic starch derived from tapioca. While this method seems to offer an affordable route for protein purification, finding a cationic starch derived from tapioca starch that meets the specifications of Catamyl-VS has proven itself impossible due to its low demand in the paper making industry here in the United States. This study aims to find a cationic starch derived from corn that is cost effective, readily available in the market, and successful in purifying MBP tagged proteins with a high degree of purity.
Four cationic starches derived from corn (Charge +99, Charge +110, Charge +309, Charge +310) were kindly gifted to us by Cargill. Prior to this study, viral fibroblast growth factor (vFGF) genes from Autographa californica M nucleopolyhedrovirus (AcMNPV) and Choristoneura fumiferana M nucleopolyhedrovirus (CfMNPV) were cloned with a coding sequence for MBP to produce plasmids coding for the fusion proteins Ac6 and Cf6, respectively. Ac6 and Cf6 plasmids were transformed into E. coli ER2508 to express the recombinant proteins. Lysate from Ac6 and Cf6 were incubated with each cationic starch at concentrations of 0.2%, 0.3%, and 0.4% (w/v) cationic starch. Precipitation was done with 10% (w/v) PEG 3350 and 50mM CaCl2. The bound MBP tagged protein was eluted off the cationic starch polymer by resuspension in 1M NaCl and separated by centrifugation. Current Bradford results show protein concentrations as high as 232µg/mL for Cf6 lysate with 0.4% (w/v) +309 cationic starch, and 311µg/mL for Cf6 lysate with 0.3% (w/v) +310 cationic starch. The Cf6 lysate with +99 and +110 cationic starches did not fall within the linear range of the BSA standard. A duplicate purification using Ac6 lysate and only +310 and +310 revealed high protein concentration in a Bradford assay, but high protein concentrations in the “No Starch” control as well. SDS-PAGE Coomassie Staining of both purification tests revealed no purification of the MBP-tagged vFGF protein and high protein precipitation in the no starch control. A lower percent PEG (6%) and lower protein concentrations were determined to run the experiment one more time to determine if we can eliminate the precipitation of unwanted proteins and purify the MBP-tagged vFGF protein. This purification is currently underway and will be followed by protein analysis, SDS-PAGE Coomassie staining, and Western Blotting. If successful, a comparison of the cationic starch purification will be compared to purification by amylose affinity chromatography. If the purification is high, I plan to continue researching the mitogenic effect vFGF has on insect and mammalian cells lines.
Comments
I wasn't able to download the original video from kaltura with video recording of myself, only the powerpoint and audio. I had to screen record the original recording to get it to work. I apologize for the lag at the beginning. I could not get it to not lag.