| 1. |
|
|
| 2. |
- Aricescu, A R, et al.
(författare)
-
Eukaryotic expression: developments for structural proteomics.
- 2006
-
Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 62, s. 1114-1124
-
Tidskriftsartikel (refereegranskat)abstract
- The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
|
|
| 3. |
- Agirre, Jon, et al.
(författare)
-
The CCP4 suite: integrative software for macromolecular crystallography
- 2023
-
Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 79, s. 449-461
-
Tidskriftsartikel (refereegranskat)abstract
- The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.
|
|
| 4. |
- Alzari, Pedro M., et al.
(författare)
-
Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE
- 2006
-
Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62, s. 1103-1113
-
Tidskriftsartikel (refereegranskat)abstract
- The implementation of high- throughput ( HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe ( SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non- ligation- based cloning techniques used, namely Gateway, ligation- indendent cloning of PCR products ( LIC- PCR) and In- Fusion, with LIC- PCR emerging as the most cost- effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library- based screening for soluble constructs and parallel small- scale high- density fermentation.
|
|
| 5. |
|
|
| 6. |
- Schulz, Sebastian A., et al.
(författare)
-
Roadmap on photonic metasurfaces
- 2024
-
Ingår i: APPLIED PHYSICS LETTERS. - 0003-6951 .- 1077-3118. ; 124:26
-
Tidskriftsartikel (refereegranskat)abstract
- Here we present a roadmap on Photonic metasurfaces. This document consists of a number of perspective articles on different applications, challenge areas or technologies underlying photonic metasurfaces. Each perspective will introduce the topic, present a state of the art as well as give an insight into the future direction of the subfield.
|
|
| 7. |
- Valegard, K, et al.
(författare)
-
Structure of a cephalosporin synthase
- 1998
-
Ingår i: NATURE. - : MACMILLAN MAGAZINES LTD. - 0028-0836. ; 394:6695, s. 805-809
-
Tidskriftsartikel (refereegranskat)abstract
- Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are cat
|
|
| 8. |
- Heidebrecht, Tatjana, et al.
(författare)
-
Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or 5-hmC : evidence for a rapid conformational change upon DNA binding
- 2012
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134:32, s. 13357-13365
-
Tidskriftsartikel (refereegranskat)abstract
- Base J (β-D-glucosyl-hydroxymethyluracil) was discovered in the nuclear DNA of some pathogenic protozoa, such as trypanosomes and Leishmania, where it replaces a fraction of base T. We have found a J-Binding Protein 1 (JBP1) in these organisms, which contains a unique J-DNA binding domain (DB-JBP1) and a thymidine hydroxylase domain involved in the first step of J biosynthesis. This hydroxylase is related to the mammalian TET enzymes that hydroxylate 5-methylcytosine in DNA. We have now studied the binding of JBP1 and DB-JBP1 to oligonucleotides containing J or glucosylated 5-hydroxymethylcytosine (glu-5-hmC) using an equilibrium fluorescence polarization assay. We find that JBP1 binds glu-5-hmC-DNA with an affinity about 40-fold lower than J-DNA (~400 nM), which is still 200 times higher than the JBP1 affinity for T-DNA. The discrimination between glu-5-hmC-DNA and T-DNA by DB-JBP1 is about 2-fold less, but enough for DB-JBP1 to be useful as a tool to isolate 5-hmC-DNA. Pre-steady state kinetic data obtained in a stopped-flow device show that the initial binding of JBP1 to glucosylated DNA is very fast with a second order rate constant of 70 μM(-1) s(-1) and that JBP1 binds to J-DNA or glu-5-hmC-DNA in a two-step reaction, in contrast to DB-JBP1, which binds in a one-step reaction. As the second (slower) step in binding is concentration independent, we infer that JBP1 undergoes a conformational change upon binding to DNA. Global analysis of pre-steady state and equilibrium binding data supports such a two-step mechanism and allowed us to determine the kinetic parameters that describe it. This notion of a conformational change is supported by small-angle neutron scattering experiments, which show that the shape of JBP1 is more elongated in complex with DNA. The conformational change upon DNA binding may allow the hydroxylase domain of JBP1 to make contact with the DNA and hydroxylate T's in spatial proximity, resulting in regional introduction of base J into the DNA.
|
|
| 9. |
- Staring, Jacqueline, et al.
(författare)
-
PLA2G16 represents a switch between entry and clearance of Picornaviridae
- 2017
-
Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 541:7637, s. 412-416
-
Tidskriftsartikel (refereegranskat)abstract
- Picornaviruses are a leading cause of human and veterinary infections that result in various diseases, including polio and the common cold. As archetypical non-enveloped viruses, their biology has been extensively studied. Although a range of different cell-surface receptors are bound by different picornaviruses, it is unclear whether common host factors are needed for them to reach the cytoplasm. Using genome-wide haploid genetic screens, here we identify the lipid-modifying enzyme PLA2G16 (refs 8, 9, 10, 11) as a picornavirus host factor that is required for a previously unknown event in the viral life cycle. We find that PLA2G16 functions early during infection, enabling virion-mediated genome delivery into the cytoplasm, but not in any virion-assigned step, such as cell binding, endosomal trafficking or pore formation. To resolve this paradox, we screened for suppressors of the ΔPLA2G16 phenotype and identified a mechanism previously implicated in the clearance of intracellular bacteria. The sensor of this mechanism, galectin-8 (encoded by LGALS8), detects permeated endosomes and marks them for autophagic degradation, whereas PLA2G16 facilitates viral genome translocation and prevents clearance. This study uncovers two competing processes triggered by virus entry: activation of a pore-activated clearance pathway and recruitment of a phospholipase to enable genome release.
|
|
