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

Sreenivas, Suma; Krishnaiah, Sateesh M.; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Jayaprakash, N.; Chatterjee, Amarnath; Sastry, Kedarnath N.

doi:10.1007/s00253-014-6052-5

Cited by 21 publications

(10 citation statements)

References 21 publications

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“…maturation were reported to be the co-overexpression of KEX2 [77,78], the post-expressional in vitro maturation by recombinant KEX2 [79] or the expression of the target protein with its native SP [70]. However, none of these approaches led to an improvement in our experiments.…”

Section: Plos Onementioning

confidence: 88%

Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris

2021

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Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

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Section: Plos Onementioning

confidence: 88%

Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris

2021

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“…A few remedial measures like addition of protease inhibitors or use of protease-deficient strains have been suggested to circumvent the problem of proteolytic degradation [8,9]. In our previous study we have described the expression of recombinant two-chain Insulin glargine in P. pastoris [10]. By over expression of the Kex2 protease we were able to secrete the fully folded two-chain glargine into the medium.…”

Section: Introductionmentioning

confidence: 94%

“…2a). The construction of the glargine and the Kex2 constructs are described in our previous study [10]. Briefly, the glargine coding sequence was cloned in pPIC9K vector in frame with AOX1 promoter and mat-a signal sequence to enable secretion of the protein.…”

Section: The Gene Disruption Cassette and Expression Plasmidsmentioning

confidence: 99%

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

Sreenivas

Krishnaiah

Mohan

et al. 2016

Protein Expression and Purification

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a b s t r a c tInsulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the Cterminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the Cterminal clipped species reduced by~80%. This modification further improved the process by reducing the levels of product related impurities.

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“…Trypsin cleaves the C-terminus of both lysine and arginine residues [33]. Since the glargine B-chain has several internal arginine and lysine residues, a number of impurities are generated following trypsin treatment [45].…”

Section: Introductionmentioning

confidence: 99%

Recombinant Glargine Insulin Production Process Using Escherichia coli

Hwang

Kim

Lee

et al. 2016

Journal of Microbiology and Biotechnology

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Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into JM109. The transformed cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.

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

Cited by 21 publications

References 21 publications

Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris

Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris

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

Recombinant Glargine Insulin Production Process Using Escherichia coli

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