High-yield overproduction and purification of human aquaporins from Pichia pastoris

Al-Jubair, Tamim; Steffen, Jonas Hyld; Missel, Julie Winkel; Kitchen, Philip; Salman, Mootaz M.; Bill, Roslyn M.; Gourdon, Pontus; Törnroth-Horsefield, Susanna

doi:10.1016/j.xpro.2022.101298

Cited by 3 publications

(3 citation statements)

References 5 publications

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“…From yeast overexpressed BoPIP1;2, 0.14 mg g -1 of pure protein were obtained, and for BoPIP2;2, the yield was even higher, 0.99 mg per gram of yeast cell. These yields are consistent with the production range of 0.1-0.5 mg of pure protein per gram of yeast cell reported by Al-Jubair et al (2022) [54] .…”

Section: Discussionsupporting

confidence: 92%

Plant aquaporin reconstituted proteoliposomes as nanosystem for resveratrol encapsulation

Yepes-Molina,

Teruel,

Johanson

et al. 2023

Preprint

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Aquaporins (AQPs) are intrinsic membrane proteins responsible for facilitating water transport across biological membranes. AQPs found in plant membrane vesicles (MV) have been related to the functionality and stability of the vesicles. In this study, we focused on AQPs obtained from Brassica oleracea var. L. italica (broccoli) by the great potential for different biotechnological applications. To gain further insight into the role of AQPs in MV and advance the biotechnological applications of AQPs, we describe the heterologous overexpression of two broccoli AQPs (BoPIP1;2 and BoPIP2;2) in Pichia pastoris, resulting in the purification of both AQPs with high yield (0.14 and 0.99 mg per gram cells for BoPIP1;2 and BoPIP2;2, respectively). We reconstituted purified AQPs in liposomes to study their functionality, showing no changes in size compared to liposomes. BoPIP2;2 facilitated water transport, which was preserved for seven days at 4oC and 25ºC but not at 37oC, whereas BoPIP1;2 did not enhance water transport across the proteoliposome membrane. Additionally, BoPIP2;2 was incorporated into liposomes to encapsulate a resveratrol extract in proteoliposome vesicles, resulting in increased entrapment efficiency compared to conventional liposomes. Molecular docking identified potential binding sites for resveratrol in PIP2s, highlighting the role of AQPs in the improved entrapment efficiency of resveratrol. Moreover, a modelling study was conducted, demonstrating interactions between a plant AQP and human integrin, which may be a benefit to increase contact and internalization by the human target cells. Thus, our results suggest that AQPs-based alternative encapsulation systems can be used in specifically target biotechnological applications.

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Section: Discussionsupporting

confidence: 92%

Plant aquaporin reconstituted proteoliposomes as nanosystem for resveratrol encapsulation

Yepes-Molina,

Teruel,

Johanson

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…From yeast overexpressed BoPIP1;2, 0.14 mg g −1 of pure protein were obtained, and for BoPIP2;2, the yield was even higher, 0.99 mg per gram of yeast cell. These yields are consistent with the production range of 0.1-0.5 mg of pure protein per gram of yeast cell reported [21].…”

Section: Discussionsupporting

confidence: 90%

Brassica oleracea L. var. italica Aquaporin Reconstituted Proteoliposomes as Nanosystems for Resveratrol Encapsulation

Yepes-Molina,

Teruel,

Johanson

et al. 2024

IJMS

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Aquaporins (AQPs), membrane proteins responsible for facilitating water transport, found in plant membrane vesicles (MV), have been related to the functionality and stability of MV. We focused on AQPs obtained from broccoli, as they show potential for biotechnological applications. To gain further insight into the role of AQPs in MV, we describe the heterologous overexpression of two broccoli AQPs (BoPIP1;2 and BoPIP2;2) in Pichia pastoris, resulting in their purification with high yield (0.14 and 0.99 mg per gram cells for BoPIP1;2 and BoPIP2;2). We reconstituted AQPs in liposomes to study their functionality, and the size of proteoliposomes did not change concerning liposomes. BoPIP2;2 facilitated water transport, which was preserved for seven days at 4 °C and at room temperature but not at 37 °C. BoPIP2;2 was incorporated into liposomes to encapsulate a resveratrol extract, resulting in increased entrapment efficiency (EE) compared to conventional liposomes. Molecular docking was utilized to identify binding sites in PIP2s for resveratrol, highlighting the role of aquaporins in the improved EE. Moreover, interactions between plant AQP and human integrin were shown, which may increase internalization by the human target cells. Our results suggest AQP-based alternative encapsulation systems can be used in specifically targeted biotechnological applications.

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“…1.5 L basal salt medium (7 mM CaSO 4 , 0. ) was inoculated with 100 mL pre-culture grown overnight in YPD [9,10,20]. The medium contained 60 mL glycerol, and when it had been consumed, a feed with 50% glycerol supplemented with PTM was started, at a rate keeping the dissolved oxygen level above 20%.…”

Section: Fed-batch Cultivation and Inductionmentioning

confidence: 99%

Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry

Werin,

Hansson Wennersten,

Olsson

et al. 2024

Microb Cell Fact

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Background The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. Results The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. Conclusions The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

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High-yield overproduction and purification of human aquaporins from Pichia pastoris

Cited by 3 publications

References 5 publications

Plant aquaporin reconstituted proteoliposomes as nanosystem for resveratrol encapsulation

Plant aquaporin reconstituted proteoliposomes as nanosystem for resveratrol encapsulation

Brassica oleracea L. var. italica Aquaporin Reconstituted Proteoliposomes as Nanosystems for Resveratrol Encapsulation

Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry

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