Cloning and Expression of AaeAPO from Agrocybe aegerita to E. coli, for Studies of Structure-Function Relationships by Site-Specific Mutagenesis

So far, much has been learned from the functional and mechanistic investigation on AaeAPO. However, one of the major limitations of AaeAPO investigation comes from the slow growth of Agrocybe aegerita and the low yield of protein production. Cloning AaeAPO from its original host to E. coli, Saccharomyces cerevisiae or Aspergillus niger might be a way to produce the protein in large amounts within a short time and to perform mutations. In this chapter, we first address the successful purification of wild type AaeAPO from Agrocybe aegerita. Next, results on the isolation of apo mRNA, RT-PCR obtaining the apo cDNA, cloning and expression AaeAPO in E. coli will be reported. However, the recombinant protein from E. coli forms inclusion bodies thus has no activity. Last, several future experiments on protein reconstitution with heme and the expression of AaeAPO in fungus strains will be discussed.