LP3’s part in PPS is described in the PPS annual report to the Swedish Research Council. This report here will mainly focus on LP3 activities outside of PPS and try to highlight LP3’s local role for Lund University.

Outside PPS, LP3 continued to offer crystallization and protein crystal screening at the BioMAX beamline at MAX IV laboratory to its users. This enables non-experts in protein crystallography to use X-ray crystallography at MAX IV and pursue structure determination, as well as LP3 continued to offer services within biophysical characterization of proteins. Also, outside PPS, LP3 continued the support to the DEMAX platform of ESS and the FragMAX platform of MAX IV.
LP3 staff was in 2022 involved in both undergraduate and graduate teaching, as well as national and international conferences and networks of interest.

In general, both within and outside PPS, LP3 continued in 2022 to deliver projects to its users at the maximum of its capacities.

Wolfgang Knecht,
Manager LP3
April. 2023

Introduction

Lund Protein Production Platform (LP3) as a center 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 and characterization, including stable isotope labeled proteins, crystallization of proteins and structure determination of proteins.
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 does therefore also handle regular screening of user crystals at BioMAX and subsequent structure determination.

Since 2022, LP3 is hosting and staffing with a part of its staff the national research infrastructure Protein Production Sweden (PPS, external hompage) Lund University node.

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.

PPS

With start in 2022, the Swedish research council (VR) has granted financing for a five-year period for a new nationally distributed research infrastructure – Protein Production Sweden (PPS). Five universities (the University of Gothenburg (host), Lund University, Karolinska Institutet, KTH Royal Institute of Technology, and Umeå University) form the infrastructure and offer expert competence in various techniques of protein production. Researchers across Sweden have access via a joint hub and have the possibility to get support based on their research needs.

As the Lund node (Figure 1) LP3 provides 3 of the 8 modules of PPS. In the context of conventional protein production modules, LP3 provides protein expression in insect cells using the baculovirus expression vector system (BEVS). In addition, LP3 is responsible for the production of (per)deuterated proteins and macromolecules for neutron scattering applications in one module (4.1) and reagents to aid in structural biology at synchrotrons in another one (4.2). With the two later modules, LP3 connects seamless to its capabilities for protein crystallization, structure determination and X-ray aided fragment screening (XFS) at FragMAX. LP3 is thereby responsible within PPS for the goal to actively create Gateway environments for Max IV and the European Spallation Source ERIC (ESS).

Dr. Knecht currently also serves as the PPS deputy director in addition to his function as node director and head of LP3.

2022 was the “startup” year for PPS. Major tasks have been to establish a common PPS website with a project application form where researchers can apply for PPS services, and the set-up of an accompanying project database. Already during the first year, 105 PIs applied for 141 different projects, showing the high demand for recombinant proteins from the Swedish scientific community. During 2022 PPS has completed 105 projects and 224 protein batches were delivered, which was possible because most staff and equipment was available from the start of PPS. To spread the information about PPS among Swedish researchers, PPS has presented its capabilities at several different conferences and meetings. The director and the management group of PPS worked together to get the joint activities up and running. In addition, a steering committee was established that could approve the strategic plan and other important documents for PPS. Overall, the PPS structure has taken form during the first year and we could immediately and successfully offer project support to many researchers.

Details about LP3s part in PPS can be found in the PPS annual report to the Swedish Research Council.

Organisation

LP3 was run in 2022 by a manager (50 % FTE, senior lecturer) and 5 research engineers. In 2022, the staffing also included 2 experts (10 %) in microbial protein production and crystallization as well as hosting Dr. Z. Fisher (Head of DEMAX (ESS), assoc. lecturer at LU). Of these 5.7 FTE at LP3, 3.2 are staffing the LU PPS node.

6 visitors were associated with LP3 in 2022, not counting the staffing of the FragMAX project.

LP3 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, two 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 “plate hotels” with the capacity to store and automatically image plates. Tecan and a TTP (Dragonfly) liquid handling systems for the preparation of crystallization screens are also available. For characterization of proteins LP3 can provide differential scanning fluorimetry (nanoDSF), dynamic light scattering system (DLS) and a size exclusion chromatography (SEC) system (with integrated multi-detector module) for the measurement of absolute molecular weight, molecular size and sample composition (OMNISEC).

For more specifics on the capabilities and services of LP3 please visit our homepage.

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), one each from Faculty of Science (FoS), Faculty of Medicine (FoM), Faculty of Engineering (FoE), MAX IV laboratory and ESS (external member) and one student (vacant during 2022). The chairman is the dean or vice 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).

Deuteration and Macromolecular Crystallization (DEMAX)

The DEMAX platform of the European Spallation Source ERIC (ESS) is co-localized with LP3 since 2016. DEMAX and LP3 are collaborating to coordinate their efforts to develop cost-effective production of deuterated biomaterials (lipids and proteins) for neutron-based methods such as protein crystallography, neutron reflectometry, and small angle neutron scattering. The underlying agreement for this was renewed for the period 2021-2025.

Dr. Knecht on behalf of LP3 applied within the first VR (Swedish Research Council) call for in-kind contributions to ESS for providing deuteration lab services. This proposal was accepted by VR and ESS. LP3 is thereby among the very first Swedish in-kind contributions to ESS. Feel welcome to read more about this on the ESS homepage, (external webpage) and on the Swedish Research Council homepage (external webpage).

FragMAX (BioMAX Fragment Screening platform)

The FragMAX platform of MAX IV (external webpage) is a Swedish Research Council financed project, with LP3, AstraZeneca AB and Saromics Biostructures AB as co-applicants. The project aims at setting up high throughput X-ray fragment screening (XFS) at the BioMAX beamline with LP3 as the partner for crystallization and sample preparation (for more information please see the publication by Lima et al. in Acta Crystallographica 2020 (external website)). FragMAX has become a permanent platform within MAX IV laboratories with its “wet labs” firmly established at LP3.

Long term strategy

The long term strategy for LP3 was outlined in the annual report 2021. In short, LP3 has the ambition within PPS to become for Swedish researchers a) Preferred supplier of (per)deuterated biomolecules for neutron scattering experiments b) Preferred supplier of specialty protein reagents for X-ray crystallography c) Preferred supplier of protein expression in insect cells with the BEVS. This is still valid and aligns very well with the overall long-term strategy of PPS. Outside of PPS, LP3 strives to continue protein characterization, protein structure determination for non-experts (non-crystallographers) and as described above to support the FragMAX platform of MAX IV laboratory and the DEMAX platform of ESS.

Covid-19

The Covid-19 situation has of course also impacted on LP3 as outlined in the annual report 2021. During 2022 the situation went back to basically pre-Covid-19.

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.

For details of current services and updates, please see the LP3 homepage.

Users and projects

Some overall user statistics will be reported here. LP3’s part within PPS is described in the annual PPS report to VR. LP3’s activities outside PPS, crystallization & structure determination, biophysics and FragMAX will be described in more detail in this report below.

Overall

46 groups used LP3 in 2022 both through PPS or as a local infrastructure for areas that are outside of PPS as for example crystallization and biophysics, the FragMAX platform or DEMAX. Of these, 35 principal investigators came from LU and 11 were external. The distribution to different faculties and external users is presented in Figure 2. The development of user groups in numbers at LP3 since 2016 is shown in Figure 3. The principal investigators in the external user group come from the ESS, other Swedish universities (for example in 2022: UU, UmU, KTH, University of Oslo (Norway)) and industry/biotech (in 2022: two companies). Of the 46 users in 2022, 16 used LP3 in the context of PPS but are not separately shown here. A breakdown of LP3s users, projects and deliveries within PPS is given in PPS annual report.

Within PPS, LP3 now takes care of modules featuring projects that inherently take longer time and work within one project than most E. coli based project (that are now channeled into PPS to other nodes). The change of PI numbers might therefore be a first indicator of the change of scope of most projects handled by LP3 now.

Figure 4 presents the total distribution of 130 unique users at LP3 in the timeframe of 2016 - 2022. Compared to end of 2021 when this number was 118, this is an increase of 12 unique users.

Protein Crystallization and biophysical protein characterization

This part of LP3 is outside of PPS and had 23 user groups. A total of 371 screening plates were processed in 2022. LP3 had 12 projects with the aim of structure X-ray crystallography managed by LP3 for non-expert users.

The distribution of user groups in crystallization and the development of the number of user groups since 2016 are shown in Figure 5 and 6.

8 of the user groups above in 2022 also used LP3 or PPS for protein production.

The number of crystallization plates is presented in Figure 7, separated after projects that LP3 runs on behalf of users (non-experts in structural biology), FragMAX projects and projects at which the user sets up the plates themselves.

Figure 7 shows the establishment of a steady level of capacity for crystallization by LP3 in projects run on behalf of non-expert users, as well as the ongoing increase of volume, that is handled within FragMAX projects.

Crystal screening at the BioMAX Beamline

267 crystals from 11 projects were tested by LP3 staff at BioMAX at 10 occasions and 70 datasets collected in total. 6 of these projects were managed by LP3 for non-expert users and resulted in 220 crystals and 40 datasets. For the other 5 projects with crystals, LP3 helped expert users to test 47 crystals at the BioMAX beamline resulting in 30 datasets. Additionally, LP3 staff supported FragMAX crystallization as outlined below.

FragMAX project (BioMAX Fragment Screening platform)

In 2022 more projects could be done than 2021, primarily due to the Covid-19 situation improving. Besides internal FragMAX project campaigns, FragMAX run 4 projects via the European iNEXT discovery access program for international users and one project for a company. Later in 2022, the FragMAX postdoc moved on to a new job and his tasks were taken over by LP3 staff.

Visibility, access, outreach

LP3 presents its services, capabilities and new developments through its Lund University-based homepage and the LUCRIS infrastructure pages, as well as at meetings (see below).

LP3 participates in relevant national and international networks and societies, (e.g., Protein Production and Purification Partnership in Europe (P4EU) (external webpage) and Core Technologies for Life Sciences (CTLS) (external webpage). Since 2019, LP3 is one of the LU infrastructures affiliated to EATRIS ERIC (the European infrastructure for translational medicine, external webpage) in their small molecule platform. LP3 is also a member of the Deuteration Network DeuNet (external webpage).

Above engagements are for dissemination of LP3’s work as well as for the exchange and adoption of new ideas and methods into LP3.

As nearly every year, LP3 was contributing to undergraduate education. LP3 contributed to the Infralife PhD course ”Integrated Structural Biology”.

Presentations of LP3 and/or of PPS by LP3 staff were provided at the following occasions in 2022:

Oral presentations

LP3 and PPS

1. EUGLOHRIA SEEDING CONFERENCE, 2022-05-16 - 2022-05-17 (virtual)
2. Infrastructure day at the Department of Biology, Lund, October 2022
3. DeuNet meeting 16 – 17th November 2022 (virtual)
4. National PI-meeting (SFBBM), Gällöfsta, Sweden, 28-29 November 2022

PPS only

1. SECOND NATIONAL MEETING OF THE SWEDISH CHEMICAL SOCIETY #SWECHEM2022, 20 – 22 June 2022, Linköping
2. 25th Swedish Conference on Macromolecular Structure and Function, 17-20 June 2022, Tällberg, Sweden

Poster presentations

LP3 and PPS

1. LINXS 3rd integrative Structural Biology symposium, May 4-6 2022, Lund
2. HALOS Final Conference, June 8 2022, Lund
3. 34th MAX IV User Meeting 2022 - Collaboration in Focus - 3 Oct 2022, 12:00 - 5 Oct 2022

LP3 only

1. EUGLOHRIA workshop, 11 May 2022 (slides submitted) – (virtual)
2. 25th Swedish Conference on Macromolecular Structure and Function, 17-20 June 2022, Tällberg, Sweden

PPS only

1. Joint ESS ILL user days, Lund, 5th – 7th October

Publications

Results and/or proteins produced at the facility were used in the following 2022 publications – including accepted in 2022 (in the time 2016 – 2022, the total number of publications has thereby reached 102):

1. Bahnan W, Happonen L, Khakzad H, Kumra Ahnlide V, de Neergaard T, Wrighton S, André O, Bratanis E, Tang D, Hellmark T, Björck L, Shannon O, Malmström L, Malmström J, Nordenfelt P (2022) A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function. EMBO Molecular Medicine n/a (n/a):e16208. doi.org/10.15252/emmm.202216208
2. Bozzola T, Johnsson RE, Nilsson UJ, Ellervik U (2022) Sialic Acid 4-N-Piperazine and Piperidine Derivatives Bind with High Affinity to the P. mirabilis Sialic Acid Sodium Solute Symporter. ChemMedChem 17 (23):e202200351. doi.org/10.1002/cmdc.202200351
3. Bozzola T, Scalise M, Larsson CU, Newton-Vesty MC, Rovegno C, Mitra A, Cramer J, Wahlgren WY, Radhakrishnan Santhakumari P, Johnsson RE, Schwardt O, Ernst B, Friemann R, Dobson RCJ, Indiveri C, Schelin J, Nilsson UJ, Ellervik U (2022) Sialic Acid Derivatives Inhibit SiaT Transporters and Delay Bacterial Growth. ACS Chemical Biology 17 (7):1890-1900. doi.org/10.1021/acschembio.2c00321
4. Christensen S, Groth L, Leiva-Eriksson N, Nyblom M, Bülow L (2022) Oxidative Implications of Substituting a Conserved Cysteine Residue in Sugar Beet Phytoglobin BvPgb 1.2. Antioxidants (Basel) 11 (8). doi.org/10.3390/antiox11081615
5. Ding B-J, Wang H-L, Al-Saleh MA, Löfstedt C, Antony B (2022) Bioproduction of (Z,E)-9,12-tetradecadienyl acetate (ZETA), the major pheromone component of Plodia, Ephestia, and Spodoptera species in yeast. Pest Management Science 78 (3):1048-1059. doi.org/10.1002/ps.6716
6. Kozielski F, Sele C, Talibov VO, Lou J, Dong D, Wang Q, Shi X, Nyblom M, Rogstam A, Krojer T, Fisher Z, Knecht W (2022) Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16. RSC Chemical Biology. doi.org/10.1039/D1CB00135C
7. Li P, Hendricks AL, Wang Y, Villones RLE, Lindkvist-Petersson K, Meloni G, Cowan JA, Wang K, Gourdon P (2022) Structures of Atm1 provide insight into [2Fe-2S] cluster export from mitochondria. Nature communications 13 (1):4339. doi.org/10.1038/s41467-022-32006-8
8. Li P, Nayeri N, Górecki K, Becares ER, Wang K, Mahato DR, Andersson M, Abeyrathna SS, Lindkvist-Petersson K, Meloni G, Missel JW, Gourdon P (2022) PcoB is a defense outer membrane protein that facilitates cellular uptake of copper. Protein science 31 (7):e4364. doi.org/10.1002/pro.4364
9. Luttens A, Gullberg H, Abdurakhmanov E, Vo DD, Akaberi D, Talibov VO, Nekhotiaeva N, Vangeel L, De Jonghe S, Jochmans D, Krambrich J, Tas A, Lundgren B, Gravenfors Y, Craig AJ, Atilaw Y, Sandström A, Moodie LWK, Lundkvist Å, van Hemert MJ, Neyts J, Lennerstrand J, Kihlberg J, Sandberg K, Danielson UH, Carlsson J (2022) Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses. Journal of the American Chemical Society 144 (7):2905-2920. doi.org/10.1021/jacs.1c08402
10. Moparthi L, Sinica V, Moparthi VK, Kreir M, Vignane T, Filipovic MR, Vlachova V, Zygmunt PM (2022) The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain. Nature communications 13 (1):6113.doi.org/10.1038/s41467-022-33876-8
11. Orozco Rodriguez JM, Krupinska E, Wacklin-Knecht H, Knecht W (2022) Protein production, kinetic and biophysical characterization of three human dihydroorotate dehydrogenase mutants associated with Miller syndrome. Nucleosides, nucleotides & nucleic acids:1-19. doi.org/10.1080/15257770.2021.2023749
12. Orozco Rodriguez JM, Wacklin-Knecht H, Knecht W (2022) Protein-lipid interactions of human dihydroorotate dehydrogenase and three mutants associated with Miller syndrome. Nucleosides, nucleotides & nucleic acids:1-22. doi.org/10.1080/15257770.2022.2039393
13. Orozco Rodriguez JM, Wacklin-Knecht HP, Clifton LA, Bogojevic O, Leung A, Fragneto G, Knecht W (2022) New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition. Int J Mol Sci 23 (5). doi.org/10.3390/ijms23052437
14. Palica K, Vorácová M, Skagseth S, Andersson Rasmussen A, Allander L, Hubert M, Sandegren L, Schrøder Leiros H-K, Andersson H, Erdélyi M (2022) Metallo-β-Lactamase Inhibitor Phosphonamidate Monoesters. ACS Omega 7 (5):4550-4562.doi.org/10.1021/acsomega.1c06527
15. Palm F, Chowdhury S, Wettemark S, Malmström J, Happonen L, Shannon O (2022) Distinct Serotypes of Streptococcal M Proteins Mediate Fibrinogen-Dependent Platelet Activation and Proinflammatory Effects. Infect Immun 90 (2):e0046221.doi.org/10.1128/iai.00462-21
16. Pimkova K, Jassinskaja M, Munita R, Ciesla M, Guzzi N, Cao Thi Ngoc P, Vajrychova M, Johansson E, Bellodi C, Hansson J (2022) Quantitative analysis of redox proteome reveals oxidation-sensitive protein thiols acting in fundamental processes of developmental hematopoiesis. Redox Biology 53:102343. doi.org/10.1016/j.redox.2022.102343
17. Vergani S, Muleta KG, Da Silva C, Doyle A, Kristiansen TA, Sodini S, Krausse N, Montano G, Kotarsky K, Nakawesi J, Åkerstrand H, Vanhee S, Gupta SL, Bryder D, Agace WW, Lahl K, Yuan J (2022) A self-sustaining layer of early-life-origin B cells drives steady-state IgA responses in the adult gut. Immunity 55 (10):1829-1842.e1826.doi.org/10.1016/j.immuni.2022.08.018
18. Wieske LHE, Bogaerts J, Leding AAM, Wilcox S, Andersson Rasmussen A, Leszczak K, Turunen L, Herrebout WA, Hubert M, Bayer A, Erdélyi M (2022) NMR Backbone Assignment of VIM-2 and Identification of the Active Enantiomer of a Potential Inhibitor. ACS Medicinal Chemistry Letters 13 (2):257-261. doi.org/10.1021/acsmedchemlett.1c00635
19. Wilson LFL, Dendooven T, Hardwick SW, Echevarría-Poza A, Tryfona T, Krogh K, Chirgadze DY, Luisi BF, Logan DT, Mani K, Dupree P (2022) The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis. Nature communications 13 (1):3314.doi.org/10.1038/s41467-022-31048-2
20. Wernersson S, Birgersson S, Akke M (2022) Cosolvent Dimethyl Sulfoxide Influences Protein–Ligand Binding Kinetics via Solvent Viscosity Effects: Revealing the Success Rate of Complex Formation Following Diffusive Protein–Ligand Encounter. Biochemistry 62 (1):44-52.doi.org/10.1021/acs.biochem.2c00507

In 2020, we started also to list deposition of data at the Protein Data Bank (PDB) with relation to LP3 and for 2022 see these 3 PDB entries: 7Z1U, 7ZOS and 7QU4.

Collaboration

We collaborate in various ways with wider society and organizations within and outside LU. In 2022 LP3 hosted 6 visitors. LP3s efforts for visibility, access and outreach are described in the equally named chapter above. LP3 has become of part of PPS, as explained before which results in numerous collaborations between the different nodes and platforms in PPS. LP3 also participated in an application within the thematic collaborative initiative call of LU addressing pandemic spread with the title “Pandemics and Alertness”, which was granted and will start up in 2023.