Studies On Crystallization Conditions Of RHSA And HSA Fusion Protein In Pichia Pastoris

Human serum albumin (HSA) is the most abundant protein in human blood plasma,made of about half of protein mass in blood. It is stable and has an extraordinarily longhalf-life in blood circulation and relatively easy to purify. In contrast, many therapeuticmolecules have short half life and are rapidly lost from the circulation because of smaller thanthe renal filtration threshold, thereby limiting their therapeutic potentials. Nowadays, somesmall molecular drugs are fused with HSA to extend their half-life, and there are differentsystems to express HSA fusion proteins. It is not clear that which HSA fusion protein can turninto a medication and which expression system is suitable for the production of HSA fusionproteins. The analysis of crystal structure of HSA fusion proteins can help us make choice.Therefore，it is necessary to study the crystallization conditions of HSA fusion proteins.Pichia Pastorios expression system was used in this study. The preparation technologyand crystallography of rHSA and rHSA-IFNα2b fusion protein were studied. The mainconclusions of the study were as follows:（1）This study established the preparation technology of rHSA through fermentationand purification technology: The engineering Pichia Pastorios GSll5/pPIC9K-rHSA wasfermented by50L fermentor. Highly purified rHSA was separated from fermentationsupernatant by ultra filter, Blue affinity chromatography, Phenyl hydrophobic chromatographyand SP ion exchange chromatography．Eventually the purity of rHSA can reach more than95%.（2）Screening kits of Hampton and EBS were used to screen crystallization condition ofrHSA, five conditions with precipitant of PEG1500, PEG3350, PEG6000, MPEG2000andMPEG5000were obtained. The crystallization condition was hydrophobic and without strongreductant like DTT. Crystals of rHSA with precipitant of MPEG2000were best for X raydiffraction, the resolution wwas3.4. Crystal structure of rHSA was analysed by moleculardisplacement method, and the crystal structure of rHSA and pHSA were same as a wholethrough alignment, whith provided reliable basis and foundation for the clinical application ofrHSA and crystal structure study of HSA fusion proteins.（3）This study established the preparation technology of rHSA-IFNα2b throughfermentation and purification technology: The engineering Pichia PastoriosGSll5/pPIC9K-rHSA-IFNα2b was fermented by50L fermentor. Highly purifiedrHSA-IFNα2b was separated from fermentation supernatant by ultra filter, Blue affinitychromatography, Octyl hydrophobic chromatography, Q ion exchange chromatography andSP ion exchange chromatography．Eventually the purity of rHSA-IFNα2b can reach morethan95%.（4）Screening kits of Hampton and EBS were used to screen crystallization condition ofrHSA-IFNα2b, three conditions were obtained, and the whole conditions were hydrophobic,which was consistent with the previous conclusion that the crystallization condition of HSAfusion proteins should be hydrophobic. Thus this is the guidance for screening crystallizationcondition of HSA fusion proteins. In addition, through screening crystallization condition of rHSA-IFNα2b on the basis of rHSA crystallization conditions, the crystal of rHSA-IFNα2bwas obtained with the precipitation of MPEG2000, and single regular crystal ofrHSA-IFNα2b was achieved through optimized the condition by seeding method. It isconvenient for the future crystal structure study of rHSA-IFNα2b. And it can be concludedthat we can not only take the screening kits but also screen on the basis of rHSAcrystallization conditions when screening the crystallization condition of HSA fusion proteins,which provide more possibility to get the crystal of HSA fusion protein.