Native-State Stability Determines the Extent of Degradation Relative to Secretion of Protein Variants from Pichia pastoris 英文参考文献.docVIP

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Native-State Stability Determines the Extent of Degradation Relative to Secretion of Protein Variants from Pichia pastoris 英文参考文献.doc

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Native-State Stability Determines the Extent of Degradation Relative to Secretion of Protein Variants from Pichia pastoris 英文参考文献

Native-StateStabilityDeterminestheExtentof DegradationRelativetoSecretionofProteinVariants fromPichiapastoris GrahamWhyteside1,MarcosJ.C.Alcocer2,JanetR.Kumita3,ChristopherM.Dobson3,MariaLazarou1,4, RichardJ.Pleass1,4,DavidB.Archer1* 1School of Biology, University of Nottingham, Nottingham, United Kingdom, 2School of Biosciences, University of Nottingham, Sutton Bonington, United Kingdom, 3DepartmentofChemistry,UniversityofCambridge,Cambridge,UnitedKingdom,4LiverpoolSchoolofTropicalMedicine,Liverpool,UnitedKingdom Abstract Wehaveinvestigatedtherelationshipbetweenthestabilityandsecretedyieldofaseriesofmutationalvariantsofhuman lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER- associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also showthat thelessstable variants areretainedwithin thecellandmay also be targetedfor degradation. Toexplore the relationshipbetweenstabilityandsecretionfurther,twodifferentsingle-chain-variable-fragment(scFv)antibodieswerealso expressedinP.pastoris,butonlyoneofthescFvsgaverisetosecretedprotein.Thenon-secretedscFvwasdetectedwithin thecellandtheUPRindicatorswerepronounced,astheywereforthepoorly-secretedHuLvariants.Thenon-secretedscFv wasmodifiedbychangingeithertheframeworkregionsorthelinkertoimprovethepredictedstability ofthescFvand secretionwasthenachievedandthelevelsofUPRindicatorswereloweredOurdatasupportthehypothesisthatlessstable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general. Citation: Whyteside G, Alcocer MJC, Kumita JR, DobsonCM, Lazarou M, et al.(2011)Native-State Stability Determin