Construction And Purification Of Recombinant GOD In Pichia Pastorius

In recent years,glucose oxidase (abbreviated GOD) uses more and more,demand is also increasing.At present,the main production fermentation using intracellular expression system,most of which exist high production costs,output high enough protein purity.low yields and other issues.Thus,with the help of genetic engineering to build excellent GOD producing strains,and it effectively high expression of great significance.In this thesis,Pichia pastoris (strain GS115) as an expression strain.Pichia pastoris expression system the expression of intracellular and secreted into the extracellular two ways,simply used to facilitate separation of the fermentation medium and purified,can be used as a general carbon methanol strictly regulate the expression of exogenous genes.Therefore,in the present experiment identified by restriction enzyme SacI saved eukaryotic expression plasmid pPIC9K-His-GOD correct recombinant plasmid pPIC9K-His-GOD was amplified transformed into E. coli,extracted and purified.pPIC9K-His-GOD enzyme with the Blg Ⅱ linearization, electric shock transformed Pichia pastoris GS115, in order to get a high secretion easily purified yeast strain engineering,nuclear DNA extraction for PCR amplification to determine whether the conversion was successful.The resulting strain expressing the fermentation,SDS-PAGE protein expression detectedThe results obtained in this thesis are:1A recombinant plasmid pPIC9K-His-GOD transform E. coli:Select endonuclease enzyme SacI linearized plasmid,the yeast expression vector detection stored pPIC9K-His-GOD,loading the obtained fragment of E.coli and purified by the alkaline lysis method PEG the method to obtain high purity plasmid pPIC9K-His-GOD.2recombinant plasmid pPIC9K-His-GOD was transformed Pichia pastoris GS115: According yeast expression system,the purified recombinant plasmid pPIC9K-His-GOD linearized with Blg Ⅱ electric shock and transformed into Pichia pastoris GS115,the integration of the target gene to the Pichia genome. Genome by PCR to verify the integration method to determine the12strains with GOD gene in the genome integration.Geneticin G-418using the filter stable GOD yeast strain containing multiple copies of the gene,designated pPIC9K-GS115-GOD04.3Expression of the fermentation of Pichia pastoris GS115:According to the characteristics of Pichia pastoris,and transformants cultured using methanol as the sole carbon and energy induced to do,GOD laboratory previously stored sequence of the genome of Aspergillus niger5’end plus a six histidine encoding gene sequence,the fermentation supernatant after protein precipitation with trichloroacetic acid in acetone,the crude enzyme solution was purified using a histidine tag,simple and quick,to determine whether a small amount of intracellular purpose protein,cells were pelleted by sonication,the same detection method,in the final analysis by SDS-PAGE of the cellular extracellular proteins.