The “new” LP3 was created as a center in June 2016 …LP3 continued in 2019 to deliver projects...
Transcript of The “new” LP3 was created as a center in June 2016 …LP3 continued in 2019 to deliver projects...
1
The “new” LP3 was created as a center in June 2016 by the Faculty of Science, the
Faculty of Medicine and LTH by combining the “old” LP3, specialized in protein
production, with LU’s protein crystallization facility.
LP3 continued in 2019 to deliver projects to its users at the maximum of its capacities.
LP3 received further funding from the involved faculties and Lund University to secure
this current level of operation and accessibility.
2019 was the first full year period, LP3 could offer protein crystal screening at the
BioMAX beamline at MAX IV laboratory to its users. This enables more and more non-
experts in protein crystallography to use X-ray crystallography at MAX IV.
LP3 staff was in 2019 involved in both undergraduate and graduate teaching, as well
as national and international conferences and networks of interest to the field.
As should be clear from the pages of this annual report 2019, LP3 continues to deliver
value-adding services to Lund University researchers.
Wolfgang Knecht,
Manager LP3
Feb. 2020
Brief Facts 2019
67 users | 122 unique deliveries in 58 protein production projects | 258 protein
crystallization plates | 210 crystals tested at the BioMAX beamline & 82 datasets
collected | 9 visitors at LP3
2
Content Introduction ............................................................................................................................................ 2
Services .................................................................................................................................................. 4
Users and projects ................................................................................................................................ 4
Visibility, access, outreach .................................................................................................................. 9
Introduction
Lund Protein Production Platform (LP3) is a focal point for expertise and equipment for
the entire process chain of production, purification, characterization, crystallization of
proteins and their structure determination and refinement, or each individual step in
the chain. LP3 is a service center that offers customer-adapted protein production,
including stable isotope-labeled proteins, crystallization of proteins and structure
determination of proteins primarily for Lund University (LU), but also for the surrounding
community.
Since 2018, LP3 is part of a Block Allocation Group (BAG) proposal of Lund
researchers and has beamtime at the BioMAX beamline at MAX IV Laboratory. LP3
can therefore now also handle regular screening of user crystals at BioMAX and
subsequent structure determination. LP3 is also a knowledge center for dissemination
and exchange of new technologies and ideas within protein production and protein
crystallization.
LP3’s mission is to:
offer open service and support, primarily to researchers at LU, with protein
production, characterization and crystallization for their research projects.
be responsible for a common and open infrastructure for protein production and
crystallization, as well as to contribute actively to the interaction of LU with MAX
IV, ESS and other relevant major research facilities, networks and initiatives.
if needed, to act as LU’s node in a national infrastructure in the protein science
area.
develop competence and methods in the area of protein sciences.
serve the surrounding community (e.g. closely located large infrastructures, small
biotech etc.).
finance part of its operations (material and machine maintenance costs) by
charging user fees and to increase this part of the funding over time.
The infrastructure was run in 2019 by a manager (50 % FTE, senior lecturer) and six
research engineers of whom four where involved in protein production and two
3
primarily in protein crystallization and protein structure determination. In 2019, the
staffing also included 2 experts (10 %) in microbial protein production and
crystallization as well as Dr. Z. Fisher (Head of DEMAX (ESS), assoc. lecturer at LU).
9 visitors were associated with LP3 in 2019.
The center is fully equipped for protein production in E. coli and insect cells
(Baculovirus Expression Vector System, BEVS). This includes flow hoods for sterile
handling of cells, temperature-controlled shakers for culturing of cells (including access
to temperature controlled rooms), centrifuges, cell homogenization equipment (e.g.,
French Press and sonicators). For purification there are several chromatography
systems, including one Äkta Avant, one Äkta Pure and one Äkta Purifier System.
Equipment for SDS PAGE, Western blotting and other standard equipment for protein
characterization and enzymatic activity assays is available at the center or within close
proximity. All documentation is captured using electronic lab notebooks. For
crystallization the facility is equipped with state-of-the-art nanolitre pipetting equipment
with the capability to handle lipidic cubic phases for membrane proteins, as well as a
“plate hotel” with the capacity to store and automatically image up to 256 plates. Tecan
and a TTP (Dragonfly) liquid handling systems for the preparation of crystallization
screens are also available.
New equipment for larger scale cell disruption was ordered in 2019, as well as
replacement of existing equipment for small volume cell disruption installed.
For more specifics on the capabilities and services of LP3, please see “Services
below”.
Placement of the infrastructure: LP3 is placed at the Biology Department (Biology
Building A, Sölvegatan 35, 22362 Lund), within the Faculty of Science (FoS) at LU.
LP3 is a separate entity within the existing administrative structure of the Department
of Biology and follows the working and delegation principles of the FoS.
Leadership of the infrastructure: LP3 is governed by a board of one chairman (Prof.
Anders Tunlid) and 6 members (Prof. Susanna Horsefield, Dr. Kajsa Paulsson, Prof.
Mikael Akke, Dr. Kajsa Sigfridsson Clauss, Dr. Sindra Petersson Årsköld, Ida Lunga),
one each from Faculty of Science (FoS), Faculty of Medicine (FoM), LTH, MAX IV
laboratory and ESS (external member) and one student. The chairman is the dean or
pro dean of the FoS. The daily business of the center is led by a manager (50 % FTE)
(Dr. Wolfgang Knecht). The manager is supported in his function by additional experts
(10 % FTE) (Currently Dr. Claes von Wachenfeldt (microbiological protein production
and deputy manager LP3) and Dr. Derek Logan (crystallization).
For a description of the history of LP3 and a detailed outline of the long term strategy
for LP3, please see the Annual report 2016 which is also available at www.lu.se/lp3.
4
Services
LP3 offers services for the entire process chain of production, purification,
characterization, protein crystallization and protein structure determination and
structure refinement or each individual step in the chain. LP3 can help with:
• Plasmids for protein production
• Recombinant protein production in bacterial (E. coli) or eukaryotic (insect) cells.
• Protein labeling (seleno-methionine incorporation, labeling with stable isotopes
(2H, 13C, 15N), biotinylation, phosphorylation)
• Protein purification
• High-throughput & nanovolume protein crystallization
• Microbiological growth monitoring (Bioscreen C)
• Biophysical protein characterization by Size Exclusion Chromatography (SEC),
Dynamic Light Scattering (DLS) and Differential Scanning Fluorimetry (DSF)
• Automated crystallization plate storage and imaging
• Protein structure determination and refinement:
Application for beamtime, however LP3 is part of a BAG for BioMAX.
MX data collection at synchrotron beamline (BioMAX MAX IV)
Process x-ray data and determine and refine the structures
Cryo-EM screening, LP3 is part of a BAG for SciLifeLab Cryo-EM in
Stockholm and Umeå
For details of current services and updates, please see LP3 homepage: www.lu.se/lp3
Users and projects
An overall user statistics will be reported here and a more detailed breakdown into
protein production and the crystallization part will also be presented.
Overall: 67 groups used LP3 in 2019. Of these, 55 principal investigators came from
LU and 12 were external. The distribution into different faculties and external users is
presented in Figure 1. The development of user groups at LP3 since 2016 is shown in
Figure 2. The principal investigators in the external user group come from the ESS,
other Swedish universities (e.g. in 2019: SLU, UU, KI, KTH, LiU, GU) and
industry/biotech (in 2019: one company).
5
122 unique deliveries were made in 58 protein production projects and 258 protein
crystallization plates (for 27 user groups) were processed. 9 visitors worked at LP3 for
periods between a few days to up to one year, or are still associated with LP3. 17
projects are set up for both protein production and crystallization at LP3 with the aim
of structure determination. Most of these projects are done by LP3 for non-expert users
in protein crystallography. The last is a big increase from 2017 with only 4 in this
category (2018: 13). This is certainly due to that from early 2018 on, LP3 could test
crystals for diffraction at the BioMAX beamline on behalf of LP3 users. 2019, was the
first full year, LP3 could offer this service.
FoS28%
FoM40%
LTH11%
MaxIV3%
Extern18%
Figure 1. Distribution of 67 user groups at LP3 in 2019
16 17 16 19
16 14 19
273 3
8
7
1 1
1
2
7 10
13
12
0
10
20
30
40
50
60
70
80
2016 2017 2018 2019
Figure 2. User groups at LP3
Extern
MaxIV
LTH
FoM
FoS
6
Protein Production: The table below shows the number and distribution of users in
2019. In brackets are the corresponding number given for previous years (2018, 2017,
2016).
first project in LP3
Extern 9 (11, 8, 5) 3 (6, 5, 5)
LTH 4 (4, 2, 2) 4 (2, 1, 0)
FoM 22 (15, 14, 14) 10 (6, 5, 6)
FoS 13 (9, 14, 12) 3 (2, 4, 5)
MAX IV 0 (1, 0, 0) 0 (1, 0, 0)
Total 48 (40, 38, 33) 20 (17, 15,16)
For 20 of the 48 principal investigators, it was the first time that they used LP3 to run a
project.
The distribution of users is illustrated in Figure 3.
FoS27%
FoM46%
LTH8%
MaxIV0%
Extern19%
Figure 3. Distribution of 48 user groups at LP3 (protein production)
7
The distribution of projects is illustrated in Figure 4.
The distribution of deliveries is illustrated in Figure 5.
Figures 3. – 5. show that within protein production about 66 – 73 % of LP3s user
groups, projects and deliveries are from and go to the FoS and FoM.
34 % and 17 % of all projects are connected to two areas of specialization of LP3,
either the BEVS or stable isotope labeling, respectively (see long term strategy in the
annual report 2016). 4 projects tested more than one expression system, meaning here
to test expression in E. coli and BEVS in parallel.
FoS29%
FoM43%
LTH9%
MaxIV0%
Extern19%
Figure 4. Distribution of 58 protein production projects at LP3
FoS28%
FoM38%
LTH7%
MaxIV0%
Extern27%
Figure 5. Distribution of 122 deliveries in protein production projects at LP3
8
Protein Crystallization: A total of 258 screening plates were processed in 2019 for 27
user groups. The distribution of user groups in crystallization and the development of
the number and distribution of user groups since 2016 are shown in Figure 6 and 7. In
contrast to protein production, here a higher percentage of users comes from the FoS
than the FoM (Figure 3 and 6).
The distribution of users of crystallography in LP3 is illustrated in Figure 6.
The distribution and number of users in protein crystallization per year during 2016 -
2019 is illustrated in Figure 7.
The sharp increase in user groups at LP3 crystallography between 2017 and 2018 can
mainly be explained by the fact, that LP3 has now projects in which LP3 does protein
FoS41%
FoM26%
LTH15%
MaxIV7% Extern
11%
Figure 6. Distribution of 27 user groups at LP3 (crystallography)
74
11 11
6
3
7 71
1
5 4
1
1
1 2
3
2
63
0
5
10
15
20
25
30
35
2016 2017 2018 2019
Figure 7 - user groups at LP3 crystallography
Extern
MaxIV
LTH
FoM
FoS
9
production and crystallization with the aim of structure determination at the BioMAX
beamline for mainly non-experts in protein crystallography. This is also visible in the
number of crystallization plates that belong to LP3 managed projects, 104 out of the
258 plates in total. This has become possible due to regular beamtime at the BioMAX
beamline and increased LP3 staffing for protein structure determination.
Also more groups use the biophysics instrumentation (DSF and DSL) at LP3
crystallography. We assume this to be a consequence of the increase of projects in
crystallization and the need for characterizing protein formulation in them as well as for
NMR and from groups getting interested in protein ligand screening.
Crystal screening at the BioMAX Beamline: 2019, was the first full year, LP3 could offer
this service. In 2019 210 crystals were tested at the BioMAX beamline and 82 datasets
collected in 15 projects. (2018: 154 crystals; 43 datasets; 13 projects).
FragMAX project (BioMAX Fragment Screening platform): A grant application in 2018
to the Swedish Research Council within the call “Grant for accessibility to
infrastructure” was successful. The main applicant was BioMAX beamline manager (U.
Müller) with LP3 manager (W. Knecht) as co-applicant, and the application also
included Saromics Biostructures AB and AstraZeneca. The application “FragMAX - a
facility for high throughput fragment screening in drug development by X-ray
crystallography” aims at setting up fragment screening at the BioMAX beamline with
LP3 as the partner for crystallization and sample preparation. In August 2019 the
project employed a postodoc, V. Talibov, to work with LP3 staff on the aspects of
sample preparation and the project did run its first 5 commissioning runs. See more at:
https://www.maxiv.lu.se/accelerators-beamlines/beamlines/biomax/user-
access/fragmax/
Visibility, access, outreach
LP3 presents its services, capabilities and new developments through Lund University-
based homepages () and the LUCRIS infrastructure pages
(https://portal.research.lu.se/portal/en/infrastructure/lund-protein-production-
platform(c98f43d2-4907-496e-a0a8-6160645cf2fc).html, as well as at meetings (see
below).
LP3 participates in relevant national and international networks and societies, (e.g.,
Protein Production Network Sweden (PPNS) (http://www.ppns.ki.se), Association of
Resources for Biophysical Research in Europe – Molecular Biophysics in Europe
(ARBRE-MOBIEU) (https://arbre-mobieu.eu), Protein Production and Purification
Partnership in Europe (P4EU) (https://p4eu.org) and Core Technologies for Life
Sciences (CTLS) (http://www.ctls-org.eu/)). Since 2019, LP3 is one of the LU
10
infrastructures affiliated to EATRIS ERIC (the European infrastructure for translational
medicine; https://eatris.eu) in their small molecule platform. LP3 became also a
member of the European Deuteration Network DEUNET (https://deuteration.net/).
In the framework of PPNS, LP3 is part of a Memorandum of Understanding between
four Swedish Universities (LU, KI, UmU, GU) with the aim to foster and encourage
collaboration and exchange of services between the four platforms involved.
This is both for dissemination of LP3’s work as well as for the exchange and adoption
of new ideas and methods into LP3. LP3 staff participates in seminar series, research
schools and conferences.
Presentations of LP3 were provided at the following occasions in 2019:
SACT, Department of Biology: Infrastructure talks (Lund, March 2019)
Advanced Isotopic Labelling Methods for Integrated Structural Biology
(Grenoble, March 2019)
HALOS Kick-off Meeting (Lund, April 2019)
SWEPROT 2020 (Tällberg, June 2019)
MAX IV Laboratory User Meeting (UM19) (Lund, September 2019)
Research School “Imaging of 3D structures” (Lund, September/October 2019)
LINXS Event: 2nd Symposium - Integrative Structural Biology (Lund, October
2019)
Protein Science Day (Lund, October 2019)
Research school QDETAILSS (Lund, October 2019)
Medicon Valley Alliance R&D network - HALOS meeting (Lund, November
2019)
Meetings/Conferences attended:
DEUNET meeting (Lund, April 2019)
ESS Science Day (Lund, May 2019)
P4EU meeting (Amsterdam, November 2019)
Results and /or proteins produced at the facility were used in the following 2019
publications:
1. Awad, W.; Al-Eryani, Y.; Ekstrom, S.; Logan, D. T.; von Wachenfeldt,
C., Structural Basis for YjbH Adaptor-Mediated Recognition of Transcription Factor
Spx. Structure (London, England : 1993) 2019, 27 (6), 923-936.e6.
2. Bagenholm, V.; Wiemann, M.; Reddy, S. K.; Bhattacharya, A.;
Rosengren, A.; Logan, D. T.; Stalbrand, H., A surface-exposed GH26 beta-
11
mannanase from Bacteroides ovatus: Structure, role, and phylogenetic analysis of
BoMan26B. The Journal of biological chemistry 2019, 294 (23), 9100-9117.
3. Boza-Serrano, A.; Ruiz, R.; Sanchez-Varo, R.; García-Revilla, J.; Yang,
Y.; Jimenez-Ferrer, I.; Paulus, A.; Wennström, M.; Vilalta, A.; Allendorf, D.; Davila,
J. C.; Stegmayr, J.; Jiménez, S.; Roca-Ceballos, M. A.; Navarro-Garrido, V.;
Swanberg, M.; Hsieh, C. L.; Real, L. M.; Englund, E.; Linse, S.; Leffler, H.; Nilsson,
U. J.; Brown, G. C.; Gutierrez, A.; Vitorica, J.; Venero, J. L.; Deierborg, T., Galectin-
3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response
in Alzheimer's disease. Acta Neuropathol 2019, 138 (2), 251-273.
4. Dahlqvist, A.; Furevi, A.; Warlin, N.; Leffler, H.; Nilsson, U. J., Stereo-
and regioselective hydroboration of 1-exo-methylene pyranoses: discovery of
aryltriazolylmethyl C-galactopyranosides as selective galectin-1 inhibitors. Beilstein J
Org Chem 2019, 15, 1046-1060.
5. Dahlqvist, A.; Mandal, S.; Peterson, K.; Håkansson, M.; Logan, D. T.;
Zetterberg, F. R.; Leffler, H.; Nilsson, U. J., 3-Substituted 1-Naphthamidomethyl-C-
galactosyls Interact with Two Unique Sub-sites for High-Affinity and High-Selectivity
Inhibition of Galectin-3. Molecules 2019, 24 (24), E4554.
6. Dahlqvist, A.; Zetterberg, F. R.; Leffler, H.; Nilsson, U. J.,
Aminopyrimidine-galactose hybrids are highly selective galectin-3 inhibitors.
Medchemcomm 2019, 10 (6), 913-925.
7. Edmunds, S. J.; Liebana-Garcia, R.; Nilsson, O.; Domingo-Espin, J.;
Gronberg, C.; Stenkula, K. G.; Lagerstedt, J. O., ApoAI-derived peptide increases
glucose tolerance and prevents formation of atherosclerosis in mice. Diabetologia
2019, 62 (7), 1257-1267.
8. Happonen, L.; Hauri, S.; Svensson Birkedal, G.; Karlsson, C.; de
Neergaard, T.; Khakzad, H.; Nordenfelt, P.; Wikstrom, M.; Wisniewska, M.; Bjorck,
L.; Malmstrom, L.; Malmstrom, J., A quantitative Streptococcus pyogenes-human
protein-protein interaction map reveals localization of opsonizing antibodies. Nature
communications 2019, 10 (1), 2727.
9. Hauri, S.; Khakzad, H.; Happonen, L.; Teleman, J.; Malmstrom, J.;
Malmstrom, L., Rapid determination of quaternary protein structures in complex
biological samples. Nature communications 2019, 10 (1), 192.
10. Kelpsas, V.; Lafumat, B.; Blakeley, M. P.; Coquelle, N.; Oksanen, E.;
von Wachenfeldt, C., Perdeuteration, large crystal growth and neutron data collection
of Leishmania mexicana triose-phosphate isomerase E65Q variant. Acta
12
crystallographica. Section F, Structural biology communications 2019, 75 (Pt 4),
260-269.
11. Koruza, K.; Lafumat, B.; Nyblom, M.; Mahon, B. P.; Knecht, W.;
McKenna, R.; Fisher, S. Z., Structural comparison of protiated, H/D-exchanged and
deuterated human carbonic anhydrase IX. Acta crystallographica. Section D,
Structural biology 2019, 75 (Pt 10), 895-903.
12. Koruza, K.; Mahon, B. P.; Blakeley, M. P.; Ostermann, A.; Schrader, T.
E.; McKenna, R.; Knecht, W.; Fisher, S. Z., Using neutron crystallography to elucidate
the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative.
Journal of structural biology 2019, 205 (2), 147-154.
13. Kumar, R.; Ignjatović, M. M.; Peterson, K.; Olsson, M.; Leffler, H.; Ryde,
U.; Nilsson, U. J.; Logan, D. T., Structure and Energetics of Ligand-Fluorine
Interactions with Galectin-3 Backbone and Side-Chain Amides: Insight into Solvation
Effects and Multipolar Interactions. ChemMedChem 2019, 14 (16), 1528-1536.
14. Kumar, R.; Peterson, K.; Misini Ignjatović, M.; Leffler, H.; Ryde, U.;
Nilsson, U. J.; Logan, D. T., Substituted polyfluoroaryl interactions with an arginine side
chain in galectin-3 are governed by steric-, desolvation and electronic conjugation
effects. Org Biomol Chem 2019, 17 (5), 1081-1089.
15. Nilsson, O.; Del Giudice, R.; Nagao, M.; Gronberg, C.; Eliasson, L.;
Lagerstedt, J. O., Apolipoprotein A-I primes beta cells to increase glucose stimulated
insulin secretion. Biochimica et biophysica acta. Molecular basis of disease 2019,
165613.
16. Pal, K. B.; Mahanti, M.; Leffler, H.; Nilsson, U. J., A Galactoside-Binding
Protein Tricked into Binding Unnatural Pyranose Derivatives: 3-Deoxy-3-Methyl-
Gulosides Selectively Inhibit Galectin-1. Int J Mol Sci 2019, 20 (15), 3786.
17. Rozman Grinberg, I.; Berglund, S.; Hasan, M.; Lundin, D.; Ho, F. M.;
Magnuson, A.; Logan, D. T.; Sjöberg, B.-M.; Berggren, G., Class Id ribonucleotide
reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes
on metal loading. JBIC Journal of Biological Inorganic Chemistry 2019, 24 (6), 863-
877.
18. Sen, B. C.; Wasserstrom, S.; Findlay, K.; Soderholm, N.; Sandblad, L.;
von Wachenfeldt, C.; Flardh, K., Specific amino acid substitutions in beta strand S2 of
FtsZ cause spiraling septation and impair assembly cooperativity in Streptomyces spp.
Molecular microbiology 2019, 112 (1), 184-198.
13
19. Stegmayr, J.; Zetterberg, F.; Carlsson, M. C.; Huang, X.; Sharma, G.;
Kahl-Knutson, B.; Schambye, H.; Nilsson, U. J.; Oredsson, S.; Leffler, H.,
Extracellular and intracellular small-molecule galectin-3 inhibitors. Scientific reports
2019, 9 (1), 2186-2186.
20. Verteramo, M. L.; Stenstrom, O.; Ignjatovic, M. M.; Caldararu, O.;
Olsson, M. A.; Manzoni, F.; Leffler, H.; Oksanen, E.; Logan, D. T.; Nilsson, U. J.;
Ryde, U.; Akke, M., Interplay between Conformational Entropy and Solvation Entropy
in Protein-Ligand Binding. Journal of the American Chemical Society 2019, 141 (5),
2012-2026.
21. Runnberg, R.; Narayanan, S.; Itriago, H.; Cohn, M., Either Rap1 or Cdc13
can protect telomeric single-stranded 3' overhangs from degradation in vitro. Scientific
reports 2019, 9 (1), 19181.