| Summary: | 碩士 === 國立交通大學 === 分子醫學與生物工程研究所 === 103 === The T7 DNA polymerase of the T7 bacteriophage is a DNA-dependent DNA polymerase. Due to T7 replication system is a simple and efficient DNA replication system, so this system model is widely used in scientific research to understand about the role of DNA molecule, DNA polymerase and accessory proteins in DNA replication system. T7 DNA polymerase has two subunit – the Gene 5 protein (gp5) encoded by bacteriophage T7 and the thioredoxin (trx) produced by host (E.coli). According to the research of scientists, the gene 5 protein of bacteriophage T3 and T7 are homologues, about 97% similarities in the amino acid sequence level.
In this paper, the research of Gene 5 protein of T3 / T7 recombinant phage was based on the T7 replication system model. The DNA sequencing data show that Gene 5 protein of T3 / T7 recombinant phage relies mainly on T3 phage DNA sequence, there are more than 99% similarity, and only four differences in the amino acid sequence.
In the results of previous experiments have discovered that the some Gene 5 mutant strains of T3 / T7 recombinant phage that affect their own replication. Therefore, the continuation of the results. I express the mutant Gene 5 protein in the E.coli, and then using DNA-affinity chromatography to analyze the DNA binding capacity of these mutant proteins. I compared the DNA binding capacity of wild-type Gene 5 protein with mutant Gene 5 proteins by DNA-affinity chromatography and found that DNA binding capacity of some mutant Gene 5 proteins are definitely different from wild type.
I employed DNA sequencing and 3D model comparison to predict which residues are important for Gene 5 protein to bind DNA. Through the T7 DNA polymerase 3D structural model comparison found that the amino acid residues at positions 364 to 367 of Gene 5 protein are very close to the bound DNA. And in accordance with the results of the DNA affinity chromatography, maybe the amino acid residues at positions 364 to 367 of Gene 5 protein are the key region for interaction with DNA.
I successfully express the wild-type Gene 5 protein of T3 / T7 phage and mutant Gene 5 protein in E. coli , and purified Gene 5 protein. And I analyzed the interaction of these mutant Gene 5 proteins with DNA. I hope these research data are useful in understanding that effects of these mutant positions on the DNA polymerase in the future.
|
|---|
