Abstract
Fibroblast growth factors (FGF's) are signaling molecules (cytokines) secreted from specific cells known as fibroblasts. They are essential for embryonic development, function postnatally in response to injury, in regulation of electrical excitability of cells, and as hormones that have a role in regulating metabolism. Viral Fibroblast Growth factors Ac6 and Cf6 have been encoded into E. coli plasmids which produce high concentrations of our target vFGFs which are then purified for testing. The vFGFs were expressed with maltose binding protein (MBP) tagged on the N-termini. MBP is used as a pseudo-chaperone with a hydrophobic cleft that can refold the protein in case of a misfold, increasing the amount of target soluble proteins from E. coli. The target protein was purified from all other soluble products by running the MBP attached to the virally encoded cytokine through an amylose resin column. The column sticks the MBP + vFGFs to the beads while all other substances run through. The MBP+ vFGFs were then removed from the amylose resin with an elution buffer into thirty microfuge tubes for each.Ac6 and Cf6. A Bradford protein assay was performed to quantify the protein yields for Ac6 and Cf6. The Ac6 growth factor yield ranged from 600 ng -2250 ngwhile the Cf6 growth factor yield ranged from 600 ng - 1200ng. Ac6 tends to have a higher yield than Cf6. Using Coomassie stained gels and western blotting techniques it is possible to find where, how much, and how pure the vFGF is. The Coomassie stained gel showed our target protein with the attached MBP along with some denatured proteins was secluded from other soluble proteins. The western blot further confirmed that our target protein was purified from other products using antibody tagging. It was found that inoculating 2 ng/mL-20ng/mL of purified vFGF onto NIH/3T3 cells in a 96-well plate resulted in increased cell proliferation and mitogenesis.
College
College of Science & Engineering
Department
Biology
Campus
Winona
First Advisor/Mentor
Casey Finnerty
Start Date
4-19-2023 9:00 AM
End Date
4-19-2023 10:00 AM
Presentation Type
Poster Session
Format of Presentation or Performance
In-Person
Session
1a=9am-10am
Poster Number
26
Included in
vFGF Protein Purification, Expression, and Proliferation Assay
Fibroblast growth factors (FGF's) are signaling molecules (cytokines) secreted from specific cells known as fibroblasts. They are essential for embryonic development, function postnatally in response to injury, in regulation of electrical excitability of cells, and as hormones that have a role in regulating metabolism. Viral Fibroblast Growth factors Ac6 and Cf6 have been encoded into E. coli plasmids which produce high concentrations of our target vFGFs which are then purified for testing. The vFGFs were expressed with maltose binding protein (MBP) tagged on the N-termini. MBP is used as a pseudo-chaperone with a hydrophobic cleft that can refold the protein in case of a misfold, increasing the amount of target soluble proteins from E. coli. The target protein was purified from all other soluble products by running the MBP attached to the virally encoded cytokine through an amylose resin column. The column sticks the MBP + vFGFs to the beads while all other substances run through. The MBP+ vFGFs were then removed from the amylose resin with an elution buffer into thirty microfuge tubes for each.Ac6 and Cf6. A Bradford protein assay was performed to quantify the protein yields for Ac6 and Cf6. The Ac6 growth factor yield ranged from 600 ng -2250 ngwhile the Cf6 growth factor yield ranged from 600 ng - 1200ng. Ac6 tends to have a higher yield than Cf6. Using Coomassie stained gels and western blotting techniques it is possible to find where, how much, and how pure the vFGF is. The Coomassie stained gel showed our target protein with the attached MBP along with some denatured proteins was secluded from other soluble proteins. The western blot further confirmed that our target protein was purified from other products using antibody tagging. It was found that inoculating 2 ng/mL-20ng/mL of purified vFGF onto NIH/3T3 cells in a 96-well plate resulted in increased cell proliferation and mitogenesis.
