Graduation Year

2016

Location

Center for Natural Sciences, Illinois Wesleyan University

Start Date

18-4-2015 9:00 AM

End Date

18-4-2015 10:00 AM

Description

Bacteriochlorophyll plays an essential role in the process of photosynthesis in photosynthetic bacteria, but several of the enzymes involved in the synthesis of this tetrapyrrole are yet to be entirely understood. The step in which the ring structure of the tetrapyrrole is formed is catalyzed by the enzyme Mg-protoporphyrin IX monomethyl ester cyclase (MPE-cyclase) which converts the substrate MPE into protochlorophyllide (Pchlide) and incorporates an oxygen atom from water. The gene bchE has been suggested to encode a protein required for MPE-cyclase activity in the photosynthetic bacterium Rhodobacter capsulatus. In order to study the cyclase enzyme, we attempted to isolate the polypeptide encoded by bchE by first expressing the protein using pRho expression vectors in R. capsulatus. With column chromatography we hoped to isolate the BchE protein for further studies and co-purify any strongly associated partners. A MSMS analysis of the elutions from the chromatography column revealed that Pyruvate carboxylase was purified along with the two propionyl-CoA carboxylase subunits alpha and beta. These results indicated that biotin from the RC-V media had out-competed the StrepII-tag of BchE for binding to the streptactin column thus leading to the purification of biotin utilizing enzymes, but confirmed that it is possible to co-purify protein partners with strong binding affinities.

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Apr 18th, 9:00 AM Apr 18th, 10:00 AM

Expression and Isolation of the BchE Encoded Protein of Rhodobacter capsulatus in Rhodobacter capsulatus

Center for Natural Sciences, Illinois Wesleyan University

Bacteriochlorophyll plays an essential role in the process of photosynthesis in photosynthetic bacteria, but several of the enzymes involved in the synthesis of this tetrapyrrole are yet to be entirely understood. The step in which the ring structure of the tetrapyrrole is formed is catalyzed by the enzyme Mg-protoporphyrin IX monomethyl ester cyclase (MPE-cyclase) which converts the substrate MPE into protochlorophyllide (Pchlide) and incorporates an oxygen atom from water. The gene bchE has been suggested to encode a protein required for MPE-cyclase activity in the photosynthetic bacterium Rhodobacter capsulatus. In order to study the cyclase enzyme, we attempted to isolate the polypeptide encoded by bchE by first expressing the protein using pRho expression vectors in R. capsulatus. With column chromatography we hoped to isolate the BchE protein for further studies and co-purify any strongly associated partners. A MSMS analysis of the elutions from the chromatography column revealed that Pyruvate carboxylase was purified along with the two propionyl-CoA carboxylase subunits alpha and beta. These results indicated that biotin from the RC-V media had out-competed the StrepII-tag of BchE for binding to the streptactin column thus leading to the purification of biotin utilizing enzymes, but confirmed that it is possible to co-purify protein partners with strong binding affinities.