Of Pha Prokaryotic Expression Vector And Functional Analysis

To indetify the function of phaA, phaB and phaC, three genes were amplifed from the subclone of pseudomanas sp. producing PHAs (Poly- 3 -hydroxyalkanoates) by PCR. PCR products were inserted into pBV-220 with double digestion of restriction enduonuclease. The expression vectors of pBV-A pBV-B and pBV-C were constructed by orientaional cloning. Through SDS-PAGE, bioactivity and function analysis of expressed protein, the function of phaA, phaB and phaC was confirmed. The results were as follows:1. Using primes designed according to the sequence of three extracted ORFs (openig reading frame). phaA, phaB and phaC were amplifed from the subclone of pseudomanas sp. producing PHA by PCR. The Gel Electrophoresis analysis showed that the molecular weights of cloned phaA, phaB and phaC were equal to fragment speculated from three ORFs.2. phaA, phaB and phaC were inserted to pBV-220 with double digest of restriction enduonuclease. The expression vectors of pBV-A, pBV-B and pBV-C were constructed by orientaional cloning. Indefication of expression vector with restriction enduonuclease digest showed that phaA phaB and phaC were in right ORFs.3. The analysis of expressed protein in different time showed that the exoteric gene began to express after 2 hour and the quantity of the protein increased with the time increase. But the quantity of the protein increase slowly when the exoteric gene was induced after 4 hour. The result showed that 4 hour was a proper time.4. The expressed result of pBV-A, pBV-B and pBV-C showed that the expressed protein was soluble and no inclusion body was been found. SDS-PAGE analysis show the molecular weight of protein expressed by phaA was 42kDa which was equal to 3-ketohilase , the molecular weight of protein expressed by phaB was 26kDa which was equal to acetoacetyl-CoA reductase, and the molecular weight of protein expressed by phaC was 63kDa which was equal to PHA synthase.5. Adding the acetyl -CoA to the protein mixture of phaA, phaB and phaC, the mixture was analysed with the wavelength of 230-240nm. The results showed that the absorption peak for PHA could be found at the wavelength of 235nm. From all facts given above, it could be confirmed that three cloned genes have bioactivity and biological function synthesizing PHA.