Annual Report and Publications

Protein Production Sweden (PPS) has in its third year consolidated its position as a national infrastructure. Project applications are entered through the common web application form into our project database and are then discussed in the management group and distributed to the different labs. 

In this third year, 145 PIs applied for 206 different projects (corresponding to 12 and 5% increase respectively compared to 2023), a smaller increase than from 2022 to 2023. Nevertheless, this shows the continued high demand for recombinant proteins from the Swedish scientific community. 

During 2024, 212 projects were completed, resulting in the delivery of 373 protein batches (15 % increase compared to 2023) showing that our infrastructure has gained momentum, but also bringing us to the edge of our capacities. 

To continue spreading the information about PPS among Swedish researchers, we have presented our capabilities at different conferences and meetings on 22 different occasions. 

New users have come, but the third year has also seen many returning users from previous years, and PPS received again very favorable user survey results. We have fully established and implemented consensus quality analyses of our proteins. Moreover, we have worked on several activities to develop our services. 

PPS follows well the plan as given in the GanTT table.

The management group has had regular bi-weekly digital meetings throughout the year. In addition, two half-day physical meetings also took place. 

The steering committee has met four times, including one physical meeting in Stockholm. One member, Magnus Wolf-Watz in Umeå, resigned from the committee and was not replaced during 2024, leaving the committee with five members. 

The scientific advisory committee (SAC), with three members, has had one meeting in November 2024 where the upcoming application for extension of PPS was discussed and input provided. 

In September, all PPS staff met for a third staff retreat with 25 participants, in Skeppsvik outside Umeå.

Module 2 Outreach: In the third year of PPS, it is still of utmost importance to reach out with information of our existence and the services, including new developments, that we can provide. During 2024, we have reached out primarily by: 

a) Our webpage with information about PPS and the online application system has been further developed. A LinkedIn page (currently 688 followers) for PPS has been maintained during 2024. We have stopped to use X during 2024.

b) Members of the PPS management group have presented PPS at various occasions at national and international conferences, workshops as well as in educational context (for a full list, see appendix 1). The total number of oral or poster presentations was 22 in 2024.

c) Another important route for spreading the knowledge of PPS to the Swedish scientific community is through cooperation with other infrastructures. This has continued in 2024. PPS produces proteins for several SciLifeLab platforms and is associated to the Integrated Structural Biology (ISB) platform as a stakeholder. PPS has been presented at several SciLifeLab days at different sites and participated in the ISB workshop, this year with participation only from the E. coli module at KI. Two persons from PPS attended the meeting of the SciLifeLab program “Pandemic Laboratory Preparedness” in August. To connect closer to the Drug Discovery and Development platform of SciLifeLab, they were invited to present their abilities within protein production at 2024’s PPS staff retreat. Additionally, LP3, the home of PPS’ Lund node, is associated as a local research infrastructure with the SciLifeLab node in Lund. PPS is also associated with EATRIS ERIC (the European infrastructure for translational medicine) and PPS is presented on the MAXESS arena which supports and facilitates industrial use of the large-scale research infrastructures MAX IV and ESS and their eco-system.AX IV and ESS and their eco-system.

d) A user survey with 34 questions was sent out to 278 applicants and PIs who had requested or received proteins from PPS in 2024. 63 replies (23%) representing users of all submodules, showed also this year, that our users overall were very satisfied with PPS services, with a mean score of >4.79 (with 5 as the best) for services overall, the project communication, the project reporting, the project support and the protein product. The lowest score, for “time from order to delivery”, had increased from 4.2 to 4.38 this year, a somewhat increased satisfaction also with this aspect. We received several positive comments like this one “The quality and swiftness of the service is excellent - keep up the good work!”.

The three largest research areas for the use of the delivered proteins were “Biomedical research”, “Molecular biology and biochemistry” and “Structural biology” (areas may be overlapping), and the three most common methods for which the proteins had been used were “Biochemical/biophysical assays”, “Immunological assays” and “Cryo-EM.

Module 3 Expression systems:

In module 3 (with its six submodules), the main part of the support and services in protein production are performed. This includes all steps from design of the expression plasmid, culturing of cells for the actual protein production in the expression system of choice, the purification and quality control.

Module 3.1 E. coli: During 2024, the two sites in this module (PSF (KI) and PEP (UmU)) saw a continued high national interest in E. coli protein production services, with 124 applications and 237 delivered protein batches. This is in parity with 2023, even though PEP lost one staff member (100% FTE) in the protein production module 3.1 from June and was first able to fill that position in November 2024. PSF in turn recruited one more staff member in November 2023, which helped to a somewhat increased throughput during 2024. To meet increasing running and production costs, both sites have raised their user fees and continued to harmonize them. The interaction between the PSF and the PEP has resulted in knowledge transfer such as shared protocols and fruitful discussions on difficult projects. 

Module 3.2 P. pastoris: The activity of YPP has increased the third year as compared to previous years, which allowed 50% increase in personnel for sixth months. The tendency in this module is still engagement in rather long and research related projects that last for more than one year. Six proteins were delivered this year. The most pronounced example is the production of high yields of BSA, where 650 mg was delivered in Spring 2024. Furthermore, as compared to the start, routines are more defined in YPP, and tenders more clearly communicated to users before initiating projects. 

Module 3.3 Insect cells (BEVS): All three modules (module 3.1, 4.1 and 4.2) making up the Lund node share the same laboratories and instruments as well as maintenance and support functions provided by LU. In all three modules, we have successfully delivered intracellular and secreted proteins, protein complexes and membrane proteins to our users in numbers reaching the limit of our capacities (23 batches were delivered from module 3.3), despite for example having implemented methods that shorten the timeline for protein production in insect cells. Nevertheless, we did test the use of fully defined chemical media for insect cells according to the operational plan 2024.

Module 3.4 Mammalian cells: During the third year of PPS, protein production in mammalian cells has continued within the Stockholm and the Gothenburg nodes and 71 protein batches were delivered, an increase with 16% from 2023. Users from last year have returned and new ones are continuously added. All development activities in the operational plan for 2024 have been worked on, and several have been finalized. This includes establishment of a vector system to generate stable pools of clones, as a complementary system to protein production by transient transfections, and protocols for isotope-labelling.

Module 3.5 Plant cells: During 2024, the Plant Cell module continued to focus on optimizing transient protein production using Plant Cell Packs (PCPs) derived from A. thaliana cell suspension cultures. PCPs represent a new platform for recombinant protein production in plant cells. The main advantages of PCPs are i) the reduced time from clone to expressed protein, which takes about 10-14 days as compared to infection with Agrobacterium and selection of stable protein producing cell lines in Plant Cell-suspension Cultures, which takes about 6 to 8 weeks, and ii) the efficient use of PCPs as a high-throughput screening platform for protein construct testing and production. In addition, Module 3.5 continues to pursue co-cultures of A. thaliana cell suspensions and genetically engineered Agrobacterium containing the target gene(s) for infecting the plant cells. At the end of 2023 and during 2024 module 3.5 has carried out several user-initiated projects and 2 protein batches were delivered to the users.

Module 3.6 Cell-free Protein Synthesis (CFPS): The CFPS reaction setup at SNC was implemented on an Opentrons OT-2 liquid handler to enable future condition screening, system optimization work and increase overall throughput in the lab. Five user projects gave rise to 13 delivered protein batches. The focus for 2025 is to continue establishing a robust workflow for incoming projects and deliver accordingly. Future development involves setting up parallel condition screening as well as implementing automated purification in small-scale with an Agilent Bravo liquid handler.

Module 4.1 (Per)deuteration and Module 4.2 Molecular chaperones. These modules together with module 3.3 constitute the Lund node of PPS. Besides many projects in both modules, development activities in 4.1 were done according to the operational plan 2024 and our work with deuterated rich media for bacterial growth was published. (Per)deuterated proteins are essential for neutron experiments (and NMR) but far from trivial to produce. One reason is the extremely high cost for heavy water and a perdeuterated carbon source necessary during the protein expression, as well as the availability of both. 

The ESS in collaboration with module 4.1 of PPS has published a methodology for protein perdeuteration (Algal Research (2024) that reduces those costs significantly making protein perdeuteration more cost-efficient. 

Module 4.2 has successfully implemented the use of the Omnisec system (multi-detection SEC) in its workflow for protein complex preparations. 13 protein batches were delivered from module 4.1 and 8 from module 4.2 in 2024.

The common PPS project database is growing and now contains 585 projects. It has been improved during the year with more functions. 

The common project application portal on the PPS website has been re-written in a different software and now works essentially without disturbances. The contributing labs now upload project reports for each completed project, to keep all data in one place in a uniform way. Moreover, more data from performed projects are now added into the database (DNA and protein sequences, expression systems and whether production and purification were successful or not). For the PPS data to be fully searchable, we may however need to change the format of this in the future. 

The importance of making our data FAIR and useful has been discussed a lot in PPS this year, and the emerging area of AI-based predictive tools for protein expression shows the importance of this.

The work continues according to the original plan and there are no major changes.

During the third year, the steering committee for PPS has had four meetings, one of which took place in Stockholm and included visits at the SciLifeLab platforms Cryo-EM and DDD. 

The Terms and Conditions for academic use of PPS have been established and published on the web page. 

A policy for prioritization of projects when there is a high demand, has also been approved. 

The annual report for 2023 and the budget and operational plan for 2025 were also approved. 

In the fall, the upcoming application for extension has been discussed in the steering committee. 

Further questions around the collection of FAIR data in PPS have also been discussed.

The economy still follows the budget with no larger deviations. 

After the third year, 99% of the received VR funding has been used and this year 60% of the total costs have been covered by co-financing. The increase of the total costs for PPS in 2024 ended at 4.4% compared to 2023, and the user fees also showed a small increase of 3.8%. 

The user fees were however 37% higher than those budgeted for this year.

The KPIs for PPS are defined as 1) the number of applied projects, 2) the number of completed projects, 3) the number of unique PIs for all applied and 4) completed projects and finally 5) the number of protein batches (samples) that were delivered. Numbers are reported for the individual modules as well as the total number. 

In 2024, PPS got 206 project applications, and 212 projects were completed. The projects came from 145 unique PIs and 136 PIs had completed projects. 373 protein batches were delivered, an increase with 15% since 2023. All modules had project applications but module 3.5 (plant cells) only got one application this year, making the national interest in this expression technique questionable. 

The relative increase in project requests and number of users was smaller this year when compared to the increase from 2022 to 23, but we are now close to our capacity limits. 

The proportion of projects that have been performed by the users’ own universities (same affiliation for user and performing lab) was found to be 51 and 55% for applied and finished projects, respectively, continuing the small but positive trend towards a nationally distributed use of PPS. The applied and finished projects from universities that are not part of PPS were 20 and 23% respectively. 

The international recognition of PPS is similar to last year (21 applications vs 24 applications in 2023 from foreign academics). 9 project applications came from the industry sector (vs 4 in 2022 and 6 in 2023), a small but positive trend.

The list of publications for 2024 contains 36 research original research articles published by our users, compared to 35 last year. 

We now see acknowledgements to PPS in the published papers, even if some authors still acknowledge the individual platforms, probably connected to the time delay from production to publication. A full publication list is reported separately.

PPS had in December 2024 60% female employees, 43% women in the management group, 60% women in the steering group, and 67% women in the SAC. 

This quite even gender distribution continues at different levels within PPS and means no immediate need for actions. 

On the user side, PPS had 30% (33% in 2023) female users (unique PIs) that sent in 29% (35% in 2023) of the project applications, and 26% (39% in 2023) of finished projects came from women. 26% (38% in 2023) of protein batches were delivered to women. 

We have no explanation for the lower share of female users and projects, but the proportion of projects from women still falls into the span that we have presented in the Gender equality plan as the typical distribution of applicants based on project applications to VR in the areas medicine and technology (a span of 25% - 50% women as applicants). 

A risk assessment was done as part of the original application. This risk assessment is considered by us as still valid. The identified risk that qualified personnel are leaving became evident in 2024 when the most experienced person in Umeå decided to quit for another job, which affected the capacity at the Umeå node, showing the importance of offering an excellent working environment. 

Along these lines, a third staff retreat was held outside of Umeå in September 2024 and further yearly retreats are planned. 

To promote further carrier-development possibilities, like courses and relevant meetings for core facility staff, PPS signed a group membership for 2024 in the non-profit association Core Technologies for Life Sciences (CTLS) for all its staff. 

PPS has earlier decided to follow the consensus guidelines issued by The Association of Resources for Biophysical Research in Europe (ARBRE-MOBIEU) and Protein Production and Purification Partnership in Europe (P4EU) regarding quality control and the agreed methods (SDS-PAGE to determine the purity, mass spectrometry to verify the identity, and analytical SEC/SEC-MALS/OMNISEC to determine that the sample is homogeneous/monodisperse) are now routinely run for delivered purified proteins. 

This year we made an internal inventory of other biophysical techniques that we have available in our labs, and that can be used when needed, especially for internal development projects. We have also run samples together with instrument companies, to try additional biophysical techniques available, as more techniques are constantly being developed. 

For PPS, with its core business of protein production, we have however decided to implement only the consensus quality control methods mentioned above, and this is then included in the user fees.

Oral presentations of PPS:

1. MAX IV User Days, Lund, January 2024
2. Department seminar, Chalmers University of Technology, Gothenburg, February 2024
3. KI Science Park After Work, February 2024
4. Course in rapid cloning, Protein Expression & Purification, Umeå, March 2024
5. KTH Protein Science Retreat, April 2024
6. P4EU 20^th Meeting, Braunschweig, Germany, July 2024
7. Yeast Retreat, Science Faculty, Gothenburg University, August 2024
8. ISB platform (SciLifeLab) workshop, August 2024
9. Master student course “Design and Production of Biomolecules” KEM350, Gothenburg, September 2024
10. KTH Protein Science PI retreat, October 2024
11. Seminar at the Division of Applied Biochemistry, LU, Lund, October 2024
12. Presentation for the Department of Chemistry, Department Day, LU, Lund, November 2024
13. SciLifeLab Lund retreat, Lund, November 2024
14. SFBBM/KVA PI-meeting, Gothenburg, November 2024
15. ELRIG - Protein Sciences in Drug Discovery, Cambridge, UK, November 2024
16. DeuNet meeting, digital, December 2024
17. Integrated Structural Biology User Day, SciLifeLab, Stockholm (presentation by a user), December 2024

Posters or roll-up:

1. MAX IV User Days, January 2024
2. Future faculty symposium, Sahlgrenska Academy, Gothenburg, February 2024
3. 26th Swedish Conference on Macromolecular Structure and Function, Tällberg, June 2024
4. SciLifeLab Pandemic Laboratory Preparedness retreat, Stockholm, August 2024
5. Chemical Biology Centre (KBC) Days, Umeå, October 2024