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

Sreenivas, Suma; Krishnaiah, Sateesh M.; Mohan, Anil H. Shyam; Mallikarjun, Niveditha; Govindappa, Nagaraja; Chatterjee, Amarnath; Sastry, Kedarnath N.

doi:10.1016/j.pep.2015.10.002

Cited by 9 publications

(1 citation statement)

References 25 publications

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“…Boehm et al[257] developed a kex1 mutant of P. pastoris, which prevented the removal of the C-terminal lysine of endostatin, at least in fermentations up to 40 h. It was speculated that degradation observed in longer runs was caused by carboxypeptidase Y released through cell lysis. Later studies also showed reduced degradation in P. pastoris kex1 strains of hirudin[272] and insulin glargine[273]. The degradation of hirudin in wild-type P. pastoris resembled the atypical Kex1 cleavage observed by Heim et al[271] in S. cerevisiae, in that the nonbasic C-terminal residues Gln65 and Leu64 (and Tyr 63 ) were removed[272].…”

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Production of protein‐based polymers in Pichia pastoris

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A long-sought objective in material science is the development of polymers with controlled monomer sequence [1, 2]. Although progress has been made in synthetic chemistry [3], the level of control evident in natural sequential polymers such as DNA and proteins is unparalleled. These biological macromolecules feature a defined molecular size, as well as a controlled sequence of the nucleotide or amino acid monomers. Proteins fold into a three-dimensional structure defined by their primary sequence, resulting in unique properties. From 20 different amino acid monomers, nature has created an awe-inspiring wealth of different proteins, including enzymes, antibodies, and peptide hormones, as well as nonbioactive proteins with a structure-forming, rheological, or colloidal function. The latter category includes proteins such as collagen and elastin that fulfill a major role in the structure of various tissues, and silks used in animal architecture such as silkworm cocoons and spider webs (for more information see Section 9.1 of the General discussion). These proteins typically feature highly repetitive sequences with biased amino acid composition, and can often reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. The natural materials derived from them display remarkable toughness, elasticity, and other properties that have inspired material scientists to mimic them using modern protein engineering. These so-called protein-based polymers, or protein polymers for short, are produced as heterologous proteins in a suitable host, just like enzymes and other proteins, although their highly repetitive sequence, biased amino acid composition, and physicochemical properties do present additional difficulty. The genes encoding natural protein polymers are sometimes used, but more often genes are synthesized that encode simplified mimics, or even completely de novo-designed protein polymers [4-6]. Multifunctional block copolymers can be prepared by combining different polymer types into one molecule [1, 2, 7-9]. Ever since the pioneering work by Cappello and Ferrari of Protein Polymer Technologies [10, 11], Escherichia coli has by far been the most widely used production host for protein polymers. This workhorse of protein engineering is genetically very well accessible, and offers fast growth and efficient low-cost production [12]. Several other hosts have been used for the production of protein polymers, including plants, insect cells, transgenic animals, Aspergillus nidulans, Saccharomyces cerevisiae,

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Production of protein‐based polymers in Pichia pastoris

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Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris

Rinnofner

Felber

Pichler

2022

Methods in Molecular Biology

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Reduced methanol input induces increased protein output byAOX1promoter in atrans-acting elements engineeredPichia pastoris

Wang

Shi

et al. 2018

Journal of Industrial Microbiology and Biotechnology

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High oxygen consumption and heat release caused by methanol catabolism usually bring difficulties to industrial scale-up and cost for protein expression driven by methanol-induced AOX1 promoter in Pichia pastoris. Here, reduced methanol feeding levels were investigated for expression of insulin precursor in a trans-acting elements engineered P. pastoris strain MF1-IP. Insulin precursor expression level reached 6.69 g/(L supernatant) at the methanol feeding rate of 6.67 mL/(h·L broth), which was 59% higher than that in the wild-type strain WT-IP at the methanol feeding rate of 12 mL/(h·L broth). Correspondingly, the insulin precursor expression level in fermentation broth and maximum specific insulin precursor production rate was 137 and 77% higher than the WT-IP, respectively. However, oxygen consumption and heat evolution were reduced, and the highest oxygen consumption rate and heat evolution rate of the MF1-IP were 18.0 and 37.7% lower than the WT-IP, respectively.

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

Cited by 9 publications

References 25 publications

Production of protein‐based polymers in Pichia pastoris

Production of protein‐based polymers in Pichia pastoris

Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris

Reduced methanol input induces increased protein output byAOX1promoter in atrans-acting elements engineeredPichia pastoris

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