| 1. |
- Alekseenko, Alisa, et al.
(författare)
-
Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples
- 2021
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.
|
|
| 2. |
- Ampah-Korsah, Henry
(författare)
-
Exploring Unorthodox Aquaporins : Characterization of NbXIPs & AtNIPs
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- AbstractAquaporins are membrane integral proteins that facilitate the transport of waterand/or other small neutral molecules across biological membranes in cells in allforms of life. Among the subfamilies of aquaporins in higher plants, the X IntrinsicProteins (XIPs) are the most recently discovered subfamily and the leastcharacterized. However, the aromatic/arginine (ar/R) selectivity filter and theproposed substrate specificity of XIPs resemble that of some NIPs aquaporinisoforms. The aim of the studies in this thesis was to functionally and structurallycharacterize Nicotiana benthamiana XIP1;1s and Arabidopsis thaliana NIP1;1 andNIP5;1 aquaporins.By using the Pichia pastoris expression system, NbXIP1;1s, AtNIP1;1 andAtNIP5;1 were successfully expressed. NbXIP1;1s and AtNIP5;1 increased thesensitivity of P. pastoris cells to boric acid. Furthermore, the N-terminally His-taggedsplice-variant NbXIP1;1α appeared more opened to boric acid than the C-terminallyHis-tagged protein when expressed in P. pastoris. In P. pastoris spheroplasts, differences in water and glycerol permeability of NbXIP1;1α and AtNIP5;1 were observed. AtNIP5;1 was permeable to water and glycerol but NbXIP1:1α was impermeable to both substrates. NbXIP1;1α and AtNIP5;1 proteins were purified by Ni-NTA affinity chromatography. While the yield obtained for NbXIP1;1α was adequate for both functional and initial crystallization studies, the initial yield for AtNIP5;1 was only sufficient for functional characterization. The current purification scheme for AtNIP5;1 needs to be optimized to obtain sufficient amounts of protein for a comprehensive crystallization study.In a stopped-flow spectrometric analysis, NbXIP1;1α was partially permeable toboric acid in proteoliposomes. Mass spectrometric analysis revealed that thepurified NbXIP1;1α protein was phosphorylated at five amino acid residues in theN-terminal region. Results from mutational studies suggested that NbXIP1;1α maybe regulated by its N-terminal domain. It was therefore proposed that NbXIP1;1α isgated by phosphorylation. By site-directed mutagenesis, it was shown that singleamino acid substitutions L79G in helix 1 and I102H in helix 2 were sufficient torender NbXIP1;1α water permeable. Water permeable NbXIP1;1α mutants offer ameans to further probe the functional properties of NbXIP1;1α. Homology modelsof wild-type and mutant NbXIP1;1α in conjunction with functional studies suggested that T246 is the residue at the helix 5 position in the ar/R filter of NbXIP1;1α. The models also revealed a previously unrecognized orientation and interaction of the conserved arginine in the ar/R filter, which could serve as a novel guide to tune the selectivity of aquaporins.
|
|
| 3. |
- Ampah-Korsah, Henry, et al.
(författare)
-
Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable
- 2017
-
Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 17:1, s. 61-61
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Aquaporins (AQPs) are integral membrane proteins that facilitate transport of water and/or other small neutral solutes across membranes in all forms of life. The X Intrinsic Proteins (XIPs) are the most recently recognized and the least characterized aquaporin subfamily in higher plants. XIP1s have been shown to be impermeable to water but permeable to boric acid, glycerol, hydrogen peroxide and urea. However, uncertainty regarding the determinants for selectivity and lack of an activity that is easy to quantify have hindered functional investigations. In an effort to resolve these issues, we set out to introduce water permeability in Nicotiana benthamiana XIP1;1α (NbXIP1;1α), by exchanging amino acid residues of predicted alternative aromatic/arginine (ar/R) selectivity filters of NbXIP1;1α for residues constituting the water permeable ar/R selectivity filter of AtTIP2;1.RESULTS: Here, we present functional results regarding the amino acid substitutions in the putative filters as well as deletions in loops C and D of NbXIP1;1α. In addition, homology models were created based on the high resolution X-ray structure of AtTIP2;1 to rationalize the functional properties of wild-type and mutant NbXIP1;1α. Our results favour Thr 246 rather than Val 242 as the residue at the helix 5 position in the ar/R filter of NbXIP1;1α and indicate that the pore is not occluded by the loops when heterologously expressed in Pichia pastoris. Moreover, our results show that a single amino acid substitution in helix 1 (L79G) or in helix 2 (I102H) is sufficient to render NbXIP1;1α water permeable. Most of the functional results can be rationalized from the models based on a combination of aperture and hydrophobicity of the ar/R filter.CONCLUSION: The water permeable NbXIP1;1α mutants imply that the heterologously expressed proteins are correctly folded and offer means to explore the structural and functional properties of NbXIP1;1α. Our results support that Thr 246 is part of the ar/R filter. Furthermore, we suggest that a salt bridge to an acidic residue in helix 1, conserved among the XIPs in clade B, directs the orientation of the arginine in the ar/R selectivity filter and provides a novel approach to tune the selectivity of AQPs.
|
|
| 4. |
- Ampah-Korsah, Henry, et al.
(författare)
-
The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain
- 2016
-
Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7:JUNE2016
-
Tidskriftsartikel (refereegranskat)abstract
- Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.
|
|
| 5. |
- Karyolaimos, Alexandros, et al.
(författare)
-
Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening
- 2019
-
Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the periplasm of Gram-negative bacteria. In E. coli, disulfide bond containing recombinant proteins are often targeted to the periplasm by an N -terminal signal peptide that is removed once it passes through the Sectranslocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the periplasm, as can the production rate. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the periplasm.
|
|
| 6. |
- Karyolaimos, Alexandros, et al.
(författare)
-
Shaping Escherichia coli for recombinant membrane protein production
- 2018
-
Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 365:15
-
Forskningsöversikt (refereegranskat)abstract
- The bacterium Escherichia coli has been widely used for the production of both pro- and eukaryotic membrane proteins. Usually, a set of standard strains as well as different culture setups and induction regimes are screened for to enhance production yields. However, on a limited scale, E. coli strains have been isolated for recombinant helical bundle membrane protein production using both selection- and engineering-based approaches. Here, we discuss how such approaches have been used so far to shape E. coli for the production of these recombinant membrane proteins and may be used in the future to further enhance production yields.
|
|
| 7. |
- Roche, Jennifer Virginia, et al.
(författare)
-
Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5
- 2017
-
Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 292:35, s. 14636-14648
-
Tidskriftsartikel (refereegranskat)abstract
- The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser256, Ser261, Ser264, and Thr269), of which Ser256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications.
|
|
| 8. |
|
|
