Strategies to maximize expression of rightly processed human interferon α2b in Pichia pastoris

Salunkhe, Shardul S.; Soorapaneni, Sudheerbabu; Prasad, Ketaki Sabnis; Raiker, Veena A.; Padmanabhan, Sriram

doi:10.1016/j.pep.2010.02.007

Cited by 26 publications

(23 citation statements)

References 29 publications

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“…IFN2a possesses an N-terminal cysteine residue that is engaged in a disulfide bond. For leader sequences that require cleavage by Kex2, incorrect processing has been reported as cleavage is sterically hindered at this cysteine (Salunkhe et al, 2010). Using EpxL-SP, which is cleaved by signal peptidase and does not require Kex2, we obtained correctly cleaved IFN2a regardless of the N-terminal Cys (correct cleavage was confirmed by N-terminal sequencing).…”

Section: Truncated Minimal Epx1 Leader Sequences Efficiently Secrete mentioning

confidence: 80%

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

et al. 2015

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Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

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Section: Truncated Minimal Epx1 Leader Sequences Efficiently Secrete mentioning

confidence: 80%

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

et al. 2015

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“…In pharmaceutical research, P. pastoris has been used to successfully express human serum albumin (23), human interferon α2b (24) and hepatitis B virus surface antigens (25) on a large scale. Currently, heterologous expression is the main tool for the production of protein drugs, and P. pastoris is one of the attractive expression hosts due to the numerous advantages over other hosts.…”

Section: Discussionmentioning

confidence: 99%

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

Wei

Tang

et al. 2012

Mol Med Report

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Abstract. hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacⅠ and transformed into Pichia pastoris GS115 (his4, Mut + ) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His + , Mut + ) with multiple hly copies. One transformant resistant to 4.0 mg/ ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol. IntroductionHuman lysozyme (EC 3.2.1.17) is a bacteriolytic enzyme that is widely distributed in a variety of tissues and body fluids (1). It hydrolyzes preferentially the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine residues that occur in the peptidoglycan cell wall structures of certain microorganisms, particularly those of Gram-positive bacteria, and therefore appears to have a role in host defense (2). The hLY comprises 130 amino acid residues and belongs to the group of lysozymes called c-type lysozymes (3). Due to its bacteriolytic activity, it is considered to be useful for medical and food uses. There is a reemergence of uses of lysozyme in various clinical trials. The addition of lysozyme to baby formulas was proposed (4), and lysozyme had also been shown to inhibit the growth of HIV-1 in vitro. Of note, the anti-HIV activity of lysozyme is independent of its muramidase activity (5). In addition, findings of previous studies have shown that it is effective in inactivating spores of Bacillus cereus in cheese by causing high hydrostatic pressure following the addition of lysozyme (6).Currently, hLY is mainly extracted from human milk and placenta, which is restricted by many factors, such as the lack of raw materials and high cost of purification. Therefore, the hen egg-white lysozyme is the most readily available form of commercial lysozyme, since it is relatively inexpensive compared...

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“…pastoris is a non-pathogenic, methylotrophic yeast. It is a versatile microorganism capable of efficient secretion (64) and possesses the ability to produce soluble and correctly folded recombinant proteins (65). It also offers numerous benefits such as the strong expression promoter (AOX1) and the ability to stably integrate expression plasmids at a specific location (66)(67)(68).…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

“…Moreover, heterologous proteins expressed by the P. pastoris strain were able to be post-translationally modified as observed in mammalian expression systems without the use of expensive culture media and cell culture equipment, which always significantly raise the cost of the mammalian cell expression systems (67,68,72). Besides, P. pastoris-expressed proteins are free from endotoxins or pyrogens, a problem always faced in bacterial-derived proteins (64), as well as viral inclusions, a problem always faced in mammalian cell-derived proteins (73). This constitutes the expressed proteins safe for human use and suitable for clinical and therapeutic applications.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Potential combinatorial effects of recombinant atypical chemokine receptors in breast cancer cell invasion: A research perspective

2013

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Abstract. Apart from their major function in the coordination of leukocyte recruitment, chemokines, in cooperation with their receptors, have been implicated in the progression of various diseases including different types of cancer, affecting survival, proliferation and metastasis. A complex network of chemokines and receptors exists in the tumor microenvironment and affects tumor development in various ways where chemokines activate typical signalling pathways by binding to the respective receptors. The identification and characterization of a group of atypical chemokine receptors [D6, Duffy antigen receptor for chemokines (DARC), ChemoCentryx chemokine receptor (CCX-CKR) and CXCR7] which appear to use unique biochemical properties to regulate the biological activities of these chemokines, is useful in the effort to therapeutically manipulate chemokines in a broad spectrum of diseases in which these chemokines play a critical role. The aim of this review was to investigate the combinatorial effect of two reported atypical chemokine receptors, D6 and DARC, on breast cancer cell invasion to understand their role and therapeutic potential in cancer treatment. In this regard, findings of the present review should be confirmed via the construction of recombinant D6 and DARC clones as well as the expression of the respective recombinant proteins using the Pichia pastoris (P. pastoris) expression system is to be performed in a future study in order to support findings of the current review.

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Strategies to maximize expression of rightly processed human interferon α2b in Pichia pastoris

Cited by 26 publications

References 29 publications

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

Potential combinatorial effects of recombinant atypical chemokine receptors in breast cancer cell invasion: A research perspective

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