Primary Exploration Of PDI Regulating Amyloid Protein Expression In Pichia Pastoris

Pichia pastoris expressed exogenous proteins can enter protein modification processing,transport,degradation and other natural activities,and has become an ideal host for exogenous protein expression.Amyloid is a pathogenic protein of amyloid deposition disease.Due to the pathogenic cause,proteins are often misfolded during the formation of the native conformation and are also called protein conformational diseases.The common diseases of amyloid deposition include neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease,cerebral amyloid angiopathy,and type II diabetes.Human cystatin C is prone to dimer depositionin vivo during the folding process and can cause degenerative cerebral amyloid angiopathy.Chicken cystatin C is similar to human cystatin C in structure and has common physiological characteristics,but has higher thermodynamic stability than human cystatin C.As a classical amyloid model,it is often used to study the pathogenic mechanisms of protein aggregation and fibrogenesis.In the previous studies,we used Pichia pastoris to express the exogenous amyloid protein,chicken cystatin C,and we did not get the ideal expression.The reason may be that when the foreign protein synthesis requires the host to use a large number of molecular chaperones to modify,some of the external Proteins do not fold correctly and are degraded or deposited in cells,resulting in decreased expression.In this process,molecular chaperones play an important role.The molecular chaperone protein disulfide isomerase was found to be closely related to neurodegenerative amyloid diseases.In this study,recombinant Pichia pastoris strains that overexpress protein disulfide isomerase were constructed,and both intracellular and extracellular levels of exogenous chicken Cystatin C were significantly increased.And then we used RNA-seq to analyze the differences in the expression of protein Chickencystatin C between overexpressing protein disulfide isomerase strains and normal strains.373 differentially expressed genes were finally screened,of which 122 differential genes were annotated to the KEGG biological pathway,including 12 gene annotations to protein transport and catabolic pathways,21 gene annotations to protein folding sorting and degradation pathways,and 24 Genes involved in the translation of proteins and so on.And the application of real-time fluorescent quantitative PCR technology to verify the transcription of the above genes.This study will further explore the potential of Pichia pastoris as a host strain for the production of exogenous recombinant protein drugs and biological vaccine preparations.It will also seek out key regulators that affect amyloid synthesis,folding,and secretion and prevent intracellular aggregation of amyloid.Latent factors provide clues.