Sec23 and Sec24 mammalian proteins assist in the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. These COP II coat proteins trap cargo proteins for transport, potentially with direct binding to cargo protein GLUT1

Bolton, Elizabeth

North, Braden

VanDerWeide, Brayden

English

Calvin Digital Commons (Calvin University)

Calvin University Calvin Digital Commons Summer Research 2021 Jan 1st, 12:00 AM Mammalian Sec Proteins Expression and Purification Elizabeth Bolton Braden North Brayden VanDerWeide Follow this and additional works at: https://digitalcommons.calvin.edu/summer_research  Part of the Developmental Biology Commons Recommended Citation Bolton, Elizabeth; North, Braden; and VanDerWeide, Brayden, "Mammalian Sec Proteins Expression and Purification" (2021). Summer Research. 7. https://digitalcommons.calvin.edu/summer_research/2021/Posters/7 This Event is brought to you for free and open access by the University Student Scholarship at Calvin Digital Commons. It has been accepted for inclusion in Summer Research by an authorized administrator of Calvin Digital Commons. For more information, please contact digitalcommons@calvin.edu. Sec23 and Sec24 mammalian proteins assist in the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. These COP II coat proteins trap cargo proteins for transport, potentially with direct binding to cargo protein GLUT1.IntroductionConclusionsReferences• Express & purify the mammalian protein homologs Sec23 A and B, and Sec24 A and B• Determine If there is direct binding between these proteins and GLUT-1 C-terminal tailBarlowe, Charles, et al. "COPII: A membrane Coat Formed by Sec Proteins That Drive Vesicle Budding from the Endoplasmic Reticulum." Cell Press, vol. 77, 17 June 1994, pp. 895-907.Figure 5: Western Blot of PET HIS Sec24 A and 24 B. Based upon the ladder identification, Sec24 A is shown at 120 kda, confirming presence of Sec24 A (indicated by the arrow). The multiple bands in the figure suggest protein degradation. ObjectivesMammalian Sec Proteins Expression and Purification Elizabeth Bolton, Braden North, Brayden VanDerWeideCalvin University, Grand Rapids, MichiganAcknowledgementsWe would like to thank Professor Arnoys, Professor Westrate, the Calvin University Biochemistry Department, and the Bernard Klamer Biology Research FellowshipFigure 1: Formation of the COP II coat allowing for cargo protein transport. (PNAS) Findings• Confirmed that Sec 23A and B, and Sec 24A and B can be expressed in T7 E. Coli cell line despite large protein size, although protein degradation was apparent in our findings.Future Direction• Obtain larger purified samples of the Sec proteins to determine interactions with GLUT-1• Explore expression prospects in mammalian cellsFigure 3: SDS-page of Sec 24A, confirming purified expression band in lane 8 at 120 kda.• Western blot analysis tests a protein against its related antibody. This test confirms one has isolated individual protein of interest, indicated by the dense bands.  Protein Expression & Purification  SDS Page Western Blot • SDS page separates proteins by molecular weight. Single bands represent distinct proteins. Based on location in comparison to the ladder, single bands suggest but do not confirm that the protein of interest is present. 	 			 !"#$"% $&#$' &((&()*+ (,$-,)*+ &(-$-()*+ ,&,,$&#$"%  ,,!"#$"'  $	 &$  	 (Plasmid ConstructionFigure 4: Pet Sec23, a plasmid used (snap gene viewer)• Small circular DNA, called plasmids, were integrated into E. Coli to express specific Sec proteins. 1: Ladder2: Uninduced Protein Expression 3: Induced Protein Expression  4: Cell lysate5: Clarified lysate6: Purified Sec24 A7: flow through8: Buffer exchangeIn order to study a protein, it must first be produced and purified.• Transformation: Plasmid containing the gene for our protein is inserted into T7 Express strain of E. Coli• Growing: Bacteria grown in high nutrient media for several hours• Induction: Introduced plasmid activated by IPTG causing production of our protein.• Purification: Bacteria split open and cellular contents purified to isolate protein of interest using His Trap column• Analysis: Purification success analyzed by SDS-PAGE, Western Blot, and functional analysisFigure 2: Basic steps in standard protein purification protocols1 2 3 4 5 6 7 81 2  1: Ladder2: Sec24 A 

Mammalian Sec Proteins Expression and Purification

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Mammalian Sec Proteins Expression and Purification

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