Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris

Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of α–factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.

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Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris

Applied Microbiology and Biotechnology ◽

10.1007/s00253-014-6052-5 ◽

2014 ◽

Vol 99 (1) ◽

pp. 327-336 ◽

Cited By ~ 6

Author(s):

Suma Sreenivas ◽

Sateesh M. Krishnaiah ◽

Nagaraja Govindappa ◽

Yogesh Basavaraju ◽

Komal Kanojia ◽

...

Keyword(s):

Pichia Pastoris ◽

Insulin Glargine ◽

Over Expression ◽

Kex2 Protease

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Paracrystalline Aggregates of Psoriatic Keratin and Their Relation to Normal Keratin Structure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120096 ◽

1985 ◽

Vol 43 ◽

pp. 680-681

Author(s):

S. Trachtenberg ◽

P.M. Steinert ◽

B.L. Trus ◽

A.C. Steven

Keyword(s):

Cell Death ◽

Stratum Corneum ◽

Intermediate Filament ◽

Skin Disease ◽

Amorphous Matrix ◽

Terminal Differentiation ◽

Proteolytic Degradation ◽

Horny Layer ◽

Chronic Skin ◽

Chronic Skin Disease

During terminal differentiation of vertebrate epidermis, certain specific keratin intermediate filament (KIF) proteins are produced. Keratinization of the epidermis involves cell death and disruption of the cytoplasm, leaving a network of KIF embedded in an amorphous matrix which forms the outer horny layer known as the stratum corneum. Eventually these cells are shed (desquamation). Normally, the processes of differentiation, keratinization, and desquamation are regulated in an orderly manner. In psoriasis, a chronic skin disease, a hyperkeratotic stratum corneum is produced, resulting in abnormal desquamation of unusually large scales. In this disease, the normal KIF proteins are diminished in amount or absent, and other proteins more typical of proliferative epidermal cells are present. There is also evidence of proteolytic degradation of the KIF.

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