Expression of Gene Product 126 from Phage Gizmo and Creation of a Substrate

Endonucleases are enzymes which cleave DNA at specific nucleotide sequences. Homing Endonucleases (HEase) are a group of endonucleases that reside as open reading frames within self-splicing introns or inteins. After expression of the gene containing the HEase the HEase goes on to cleave a target site in a homolog of the hosting gene to induce homologous recombination turning the vacant homolog into a homing endonuclease gene (HEG). It is predicted that gene product (gp126) from the mycobacteriophage Gizmo encodes a HEase. To test whether the gene encodes a HEase the phage sequence of gp126 Gizmo was transferred into the his-tag containing plasmid (PET14b). The protein has been expressed and purified. To test the activity of the HEase a substrate is being created that will allow the predicted function to be directly tested in an enzyme assay

Functional Analysis of a Putative Homing Endonuclease

Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains such as DNA and RNA. A subgroup, homing endonucleases (HEase) are also able to propagate their encoding genes by transforming host genes to incorporate the homing endonuclease gene (HEG). This process is initiated by the expression of the HEase from the HEG, which then cleaves a homolog. The homolog subsequently undergoes a homologous recombination event as a repair mechanism, and in the process integrates a copy of the HEG. Based on predicted sequence, gene product (gp126) from the mycobacteriophage Gizmo is thought to encode for a HEase. Previous work constructed a vector for gp126 from which protein was expressed and purified. The goal of this study was to demonstrate that the DNA of the mycobacteriophage Shrimp was a viable target for the HEase and to characterize the function of the homing endonuclease. Shrimp has a very similar sequence to Gizmo, which contains the putative homing endonuclease, making it a viable substrate for experimentation. Demonstration of DNA binding will be a first step in the characterization of the interactions between gp126 and the Shrimp DNA

Expression and Isolation of the BchE Encoded Protein of \u3cem\u3eRhodobacter capsulatus in Rhodobacter capsulatus\u3c/em\u3e

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