Title of Presentation or Performance
Expression and Purification of the BciE Protein from Dinoroseobacter shibae
Major
Biochemistry
Submission Type
Poster
Area of Study or Work
Biochemistry, Biology
Expected Graduation Date
2022
Location
CNS Atrium, Easel 25
Start Date
4-9-2022 8:30 AM
End Date
4-9-2022 9:45 AM
Abstract
Photosynthesis is a prominent metabolic pathway in members of the Kingdom Plantae. This pathway is dependent upon the production of chlorophyll. Chlorophyll is a pigment molecule within the tetrapyrrole family that captures light. The Xantha-l gene in barley (Hordeum vulgare L.) encodes the Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. MPE cyclase catalyzes the formation of the isocyclic E-ring in chlorophyll biosynthesis. Previous studies have hypothesized that the BciE protein subunit is required for MPE cyclase activity in some organisms, but this has not been demonstrated in vitro. The function of the BciE protein remains unknown. The purpose of our work is to express and purify the BciE protein in large quantities. Large quantities of purified BciE protein are necessary for protein crystallization and other methods to discern the function of this protein.
The pCDFDuet-1 BciE vector (with BciE from Dinoroseobacter shibae containing a His-tag) was transformed into BL21 DE3 cells. Cell cultures were induced with 200 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) after reaching an OD600 of 0.4-0.6 in order to promote protein synthesis. Induced cultures were grown at 15oC for 67 hours. Cell culture pellets were sonicated to lyse the cells and allow for separation of the soluble and insoluble components. Soluble and insoluble components were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A band was observed at ~15 kDa, the expected molecular weight of the BciE protein. The suspected BciE polypeptide was purified through His-Tag chromatography. Fractions were subjected to another SDS-PAGE. A ~15 kDa band was observed in fraction 4, which strongly supports the presence of the BciE protein.
Through these experiments, we were able to express and purify the BciE protein. We are still working to optimize the level of expression. Variables to be tested include time of incubation, growth temperature and media conditions.
Expression and Purification of the BciE Protein from Dinoroseobacter shibae
CNS Atrium, Easel 25
Photosynthesis is a prominent metabolic pathway in members of the Kingdom Plantae. This pathway is dependent upon the production of chlorophyll. Chlorophyll is a pigment molecule within the tetrapyrrole family that captures light. The Xantha-l gene in barley (Hordeum vulgare L.) encodes the Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. MPE cyclase catalyzes the formation of the isocyclic E-ring in chlorophyll biosynthesis. Previous studies have hypothesized that the BciE protein subunit is required for MPE cyclase activity in some organisms, but this has not been demonstrated in vitro. The function of the BciE protein remains unknown. The purpose of our work is to express and purify the BciE protein in large quantities. Large quantities of purified BciE protein are necessary for protein crystallization and other methods to discern the function of this protein.
The pCDFDuet-1 BciE vector (with BciE from Dinoroseobacter shibae containing a His-tag) was transformed into BL21 DE3 cells. Cell cultures were induced with 200 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) after reaching an OD600 of 0.4-0.6 in order to promote protein synthesis. Induced cultures were grown at 15oC for 67 hours. Cell culture pellets were sonicated to lyse the cells and allow for separation of the soluble and insoluble components. Soluble and insoluble components were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A band was observed at ~15 kDa, the expected molecular weight of the BciE protein. The suspected BciE polypeptide was purified through His-Tag chromatography. Fractions were subjected to another SDS-PAGE. A ~15 kDa band was observed in fraction 4, which strongly supports the presence of the BciE protein.
Through these experiments, we were able to express and purify the BciE protein. We are still working to optimize the level of expression. Variables to be tested include time of incubation, growth temperature and media conditions.