Affinity tags can reduce merohedral twinning of membrane protein crystals

Backmark, Anna; Nyblom, Maria; Törnroth-Horsefield, Susanna; Kosinska-Eriksson, Urszula; Nordén, Kristina; Fellert, Maria; Kjellbom, Per; Johanson, Urban; Hedfalk, Kristina; Lindkvist‐Petersson, Karin; Neutze, Richard; Horsefield, Rob

doi:10.1107/s090744490802948x

Cited by 5 publications

(3 citation statements)

References 27 publications

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“…This was seen for one multi-copy HsAQP5 clone with high levels of recombinant protein expression, where 145 mg of functional protein was purified from 1 L fermenter culture (unpublished data). Protein from this clone was also used for crystallisation and rendered well ordered crystals diffracting to 3.1 Å [34,42], confirming the high quality of the expressed protein.…”

Section: Discussionmentioning

confidence: 93%

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

et al. 2011

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BackgroundWhen performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast Pichia pastoris.ResultsBy comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.ConclusionsWe report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant P. pastoris clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of P. pastoris clones with high expression levels of aquaporins and other classes of membrane proteins.

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Section: Discussionmentioning

confidence: 93%

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

et al. 2011

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“…The generation of the AQP5 constructs, membrane preparation, and protein purification have been previously described for a full-length construct with His-tag and full-length construct without His-tag [21]. The untagged truncated constructs N228Stop and T242Stop were generated using site-directed mutagenesis.…”

Section: Aqp5 Expression Purificationmentioning

confidence: 99%

Unraveling Human AQP5-PIP Molecular Interaction and Effect on AQP5 Salivary Glands Localization in SS Patients

Chivasso

Nešverová

Järvå

et al. 2021

Cells

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Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren’s syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

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“…Common methods of troubleshooting in this case may be to employ (i) a shorter (truncated) protein/domain fragment (as a flexible region, including a short histidine tag, can inhibit crystallization and may require cleaving off) or (ii) a (closely related) homologous protein from a different species for crystallization. Here, we present the crystallization of the recombinant wild-type ARF of S. solfataricus strain P1 using an alternative approach in a form suitable for X-ray studies and preliminary X-ray data analysis; although the role of a flexible affinity tag in protein crystallization has been the subject of considerable debate (Smyth et al, 2003;Carson et al, 2007;Smits et al, 2008;Backmark et al, 2008), we introduced an expression vectorderived long N-terminal extension plus a hexahistidine tag into the target protein of interest, which enabled a drastic improvement of its crystal quality. Thus, in some cases, this complementary strategy can reduce unfavourable interactions in protein crystallization and/or induce important crystal contacts and may aid a whole structuromewide robust study on a selected organism with known genomic sequence and/or a target structural study of particular proteins, e.g.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) fromSulfolobus solfataricusP1

et al. 2010

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The hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe-2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 A resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 60.72, c = 83.31 A. The asymmetric unit contains one protein molecule.

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Affinity tags can reduce merohedral twinning of membrane protein crystals

Cited by 5 publications

References 27 publications

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

Unraveling Human AQP5-PIP Molecular Interaction and Effect on AQP5 Salivary Glands Localization in SS Patients

Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) fromSulfolobus solfataricusP1

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