Expression Of Tat-Oct4in Pichia Pastoris And Study On Its Large-scale Fermentation And Purification Process

Oct4is so far discovered as one of the most significant transcription factors tomaintain pluripotency of stem cells. How well Oct4is expressed has a direct effect onpluripotency of an embryonic stem cell and its differentiation, therefore it is essentialto ESC. In2006, Japanese scientists transcribed four genes C-myc、Klf4、Oct4、Sox2into mouse fibroblasts and got induced pluripotent stem (iPS) cells. Later, morefindings showed that Oct4is the exclusive irreplaceable transcription factor in theprocess of new cell encoding but how it functions remains unknown and needs furtherresearch. Yet until now, most studies of Oct4focus on inducing Oct4and othertranscription factors into cells by viral vectors. It is a big problem in biologicalsecurity to induce heterogeneous genes into receptor cells. Consequently, it has been amajor concern on how to avoid problems caused by viral vectors and heterogeneousgenes in new cell encoding process.Tat is transactivator of transcription of HIV-Ⅰ. Covalentconnectivitycombination of Tat and heterogous protein makes heterogous protein transferred tocells and animal tissues, namely HIV-Tat contains protein transduction activity.Hikaru et al. found that the11amino-acid residues from47to57were coretransduction part in Tat protein to complete transduction and the gene sequencenumber is YGRKKRRQRRR.Tat47-57could help covalent connected DNA, protein and other molecules intoalmost all issues and cells, even through blood brain barrier with a high transductionefficiency and little damage to cells and protein bioactivity could be retained..So far, E.coli expression vector system has been used to express Tat and Oct4in some researches. However, E.coli, as prokaryotic expression system, is lack ofmodifying and processing of posttranslational expression products. Pichia pastoris, asa highly effective eukaryotic expression system, protein expression of heterogousprotein is regulated by the most accurate promoter AOX1. Besides, pichia pastoris, asMut pastoris, can use methanol as the exclusive carbon source and energy source tomodify and process posttranslational protein to keep its natural activity as much aspossible. Since pichia pastoris requires simple culture condition but grows fast, it isvery suitable for high density continuous culture with a high protein yield. Lesssecreted protein of pichia pastoris in the whole expression process has reduceddifficulty in purification. To sum up, Tat-Oct4fusion protein is suitable for large scaleproduction.In this study, we prepared large amount of recombinant human Tat-Oct4proteinsby gene engineering and fermentation engineering technology. And then we purifiedprepared proteins to establish a complete and effective purification method. Finallywe studied bioactivity of Tat-Oct4proteins. Our work includes:1. Preparation of Tat-Oct41.1Construction of the target geneBased on published human Oct4DNA sequence, Tat sequence and HSAsp inGene Bank, we designed premiers. Then RNA were extracted from human livers.RT-PCR was performed to obtain a specific DNA fragment HSAsp-Tat-Oct4.1.2Construction of and screening for expression engineering bacteriaFull length HSAsp-Tat-Oct4gene was introduced into eukaryotic expression vectorpPICZα. The recombinant pPICZα-HSAsp-Tat-Oct4was transformed into P. pastorisX-33after sequencing. Construction of expression engineering bacteria for P. pastorisTat-Oct4was done. Then, high level expressed strains were screened by SDS-PAGEanalysis. Results of Western blot analysis proved that expression of Tat-Oct4fusionprotein. Also the result of N-terminal sequencing of the expressed protein showed thesequence of the first eight amino acids was the same as expected. 1.3Optimization of Tat-Oct4Optimization process of Tat-Oct4expression in centrifuge tube(50ml) showed:the optimal induction time was144h and the optimal pH was8.0.1.4Large-scale Fermentation of Tat-Oct4Methods of Large-scale fermentation of Tat-Oct4was established based on theresult of pre-scale-up expression and optimization process, as well as control of pH,components, dissolved oxygen, formaldehyde concentration and other factors offermentor culture medium.1.5Purification of Tat-Oct4In purification of Tat-Oct4，bacteria were removed from the broth by centrifugationand supernatant was collected. Proteins were purified by vivaflow200ultrafilterconcentrations. The buffer solution was diluted first, then was separated and purifiedby chromatography. Finally, we obtained95.7%pure Tat-Oct4with38.3%recoveryNew methods of large-scale preparation and purification of Tat-Oct4have beenestablished.2. Bioactivity of Tat-Oct4We used recombinant protein Tat-Oct4to induce and culture HDFs, while addingsmall molecule VPA. Results of cell morphological observation, real-time RT-PCRand immunofluorEScence induction indicated that induced cells had some features ofstem cells. The finding provides a theoretical foundation to obtain iPS throughsingle protein.To sum up, this study has provided a new method to prepare high purifyTat-Oct4on a large scale by gene engineering technology and proved biological effectof Tat-Oct4.