Annual report 2023

Protein Production Sweden (PPS) has in its second year continued its establishment as a national infrastructure. All project applications can now be entered through the common web application form into our project database. They are then discussed in the management group and distributed to the different labs. In this second year, 137 PIs applied for 197 different projects, increases corresponding to 30% and 40% respectively, compared to 2022. This shows the continued high demand for recombinant proteins from the Swedish scientific community. During 2023, 168 projects were completed, giving rise to the delivery of 324 protein batches (47% increase compared to 2022) showing that our infrastructure has gained momentum, but also bringing us to the edge of our capacities. To continue to spread the information about PPS among Swedish researchers, we have presented our capabilities at different conferences and meetings on 24 different occasions. New users have come, but the second year has also seen many returning users from 2022, and PPS received very favorable user survey results again this year. We have agreed upon methods that ensure the quality of the delivered proteins. Moreover, we have worked on several development activities to establish more protocols that our users can now take advantage of.

PPS has followed the plan as given in the GanTT table in the application, with the exception that the planned information tours have been and will be replaced by more directed, continuous presentations at conferences, meetings, workshops, Ph.D. schools etc. as well as by continuous use of social media.

The management group has had regular bi-weekly digital meetings throughout the year. In addition, a half-day physical meeting combined with the staff meeting in September also took place. The steering committee has met four times, including one live meeting in Gothenburg. There were no changes in the composition of the management group and the steering committee, to what was described in the 2022 report. A scientific advisory committee (SAC) has been appointed and consists of Dr Tomas Lundqvist (chair, Camfil AB, Sweden), Dr Kim Remans (EMBL, Germany) and Dr Kate Smith (Glaxo Smith Kline, UK). The SAC had a first meeting after their appointment, in June, and a second meeting in October to discuss the upcoming Inventory of Research Infrastructure Needs of the Swedish research council. In September, all PPS staff met for a second staff retreat with 26 participants, outside Stockholm.

In the second year of PPS, it is still of utmost importance to reach out with information of our existence and the services that we can provide. During 2023, we have reached out primarily by: 

a) Our webpage with information about PPS and the online application system has been further developed. Work on a new website (www.pps.gu.se), that provides a presentation of PPS independent from the web format of the University of Gothenburg, has been finished and migration of the old website and new content has been done while writing this report. A LinkedIn channel (currently 525 followers) and a X (Twitter) account for PPS have been maintained during 2023.

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. The total number of presentations has increased from 14 in 2022 to 24 in 2023.

c) Another important route for spreading the knowledge of PPS to the Swedish scientific community is through cooperation with other infrastructures. This has been described in the 2022 report and was continued in 2023. PPS produces proteins for several SciLifeLab platforms and is associated to the Integrated Structural Biology platform as a stakeholder. Hanna Tegel and Malin Bäckström attended the meeting of the SciLifeLab program “Pandemic Laboratory Preparedness” in August. Additionally, LP3, the home of PPS’ Lund node, became associated as a local research infrastructure with the SciLifeLab node in Lund. In 2023, PPS also became associated with EATRIS ERIC (the European infrastructure for translational medicine). PPS is now presented on the MAXESS arena which is supporting and facilitating industrial use of the large-scale research infrastructures MAX IV and ESS and their eco-system.

d) Popular science description. PPS published a popular science description “Skördetid i proteinfabriken” (only available in Swedish) in the journal of the Swedish Chemical Society. The article is also available at the PPS webpage under news.

e) A user survey with 23 questions was sent to 180 applicants and PIs who had received proteins from PPS in 2023. 45 replies (25%) showed that the users overall were very satisfied with PPS services (>4.66 out of 5 as the best) and the scores were high (>4.55) also for project communication, reporting, project support and protein product. The lowest score (4.2) was for the time from order to delivery. Most users said they would use PPS again and recommend PPS to others. 22% of the users had stated “Biomedical research” as the use of the protein and the corresponding value was 20% for “Biochemistry/molecular biology” and 20% for “Structural biology”. As for which methods the protein had been used for, “biochemical/biophysical assays” was the most common (23%) followed by “crystallography” (12%) and “ELISA” (11%).

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 2023, the two labs in this module (PSF (KI) and PEP (UmU)) saw a continued high national interest in E. coli protein production services, with 135 applications and 233 delivered protein batches. This is a ~40% increase compared to 2022. The two sites have harmonized their user fees. The interaction between PSF and PEP has resulted in shared protocols and some shared projects on difficult proteins. In one extra- large-scale request, with totally 50-liter E. coli expression culture followed by purification, the workload was shared between PEP, PSF and the Swedish NMR center (GU).

Module 3.2 P. pastoris: The activity in the Yeast Protein Production (YPP) module has increased substantially in year two with a continued flow of requests. Intracellular, secreted as well as integral membrane proteins have been produced and growth for ¹⁵N-labelled BSA has been established. Furthermore, protocols for detergent screen as well as large scale purification have been established for recombinant proteins produced in yeast.

Module 3.3 Insect cells (BEVS): All three modules (module 3.3, 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. All modules had projects running during 2023, also fitting in relation to proportional staffing of the different modules. A method protocol of standard operating procedures for the BEVS was published (Sullivan, H. M., et al. (2023). Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS). Advanced Methods in Structural Biology. Â. Sousa and L. Passarinha. New York, NY, Springer US: 55-77) Development activities were for 3.3 done according to the operational plan 2023, in the areas of more efficient SOP, evaluation of new media for insect cell culture and implementation of a new FPLC system.

Module 3.4 Mammalian cells: During the second year of PPS, protein production in mammalian cells has continued within the Stockholm and the Gothenburg nodes and 61 protein batches were delivered. Users from last year have returned and new ones are continuously added. A new chromatography system, funded through PPS and including a set-up for analytical SEC, was implemented in the beginning of the year at the node in Gothenburg. In Stockholm, one new member of staff was recruited during the spring. All planned development activities have been worked on and some have been finalized.

Module 3.5 Plant cells: During 2023, with equipment and staff in place, the Plant Cell module focused on optimizing protein production e.g., by introducing different methods to optimize transcription and translation as well as protein secretion. We pursued two different ways of producing proteins: 1. Co-cultures of A. thaliana cell suspensions and genetically engineered Agrobacterium containing the target gene(s) with the aim of infecting the plant cells. This method takes about 5 weeks to produce protein due to a selection process for transformed plant cells. 2. Use of Plant Cell Packs (PCPs) derived from A. thaliana cell suspension cultures, which are in bulk infected Agrobacteria containing the target gene(s). The second method requires no selection and takes only about 10 days to produce the desired protein(s). Furthermore, to improve protein purification efficiency and yield, various techniques to open plant cells were evaluated and the length of the His-tag was optimized for best IMAC purification. Towards the end of the year, several user projects were initiated.

Module 3.6 Cell-free: Cell-free protein synthesis (CFPS) allows the production of diverse protein samples on demand by utilizing the cellular transcriptional and translational activities within a test tube. After optimization and adjustment of the expression system setup during 2022 and 2023, CFPS is now offered and used in ongoing PPS projects. Future enhancements will focus on automation and isotope incorporation strategies to enable NMR spectroscopy of larger protein systems.

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 projects, development activities in 4.1 were done according to the operational plan 2023 in the areas of deuterated rich media for bacterial growth resulting in a manuscript currently under revision, alternative bacterial strains for deuteration and deuteration in using eukaryotic cells. No development activities had been planned for module 4.2 in 2023.

The common PPS project database is functional and has been continuously used and improved during the year with more functions. The common project application portal on the PPS website has been up and running most of the time, but had to be completed with a PDF application form in some periods of malfunctioning of the web form. These disturbances will hopefully be removed in 2024 as the application module is presently being re-written in a different software. The contributing labs can now upload project reports for each completed project, to keep all data in one place in a uniform way.

For the data to be searchable, we may however change the format of this in the future.

The work continues according to the original plan and there are no changes besides the appointment of the SAC (Scientific Advisory Committee)

In the second year, important decisions for the steering committee have been 1) to define the roles for the SAC (Scientific Advisory Committee) and to appoint its members (mentioned above), 2) to approve the annual report for 2022 as well as the budget and operational plan for 2024 and 3) to approve a template for the agreement for access to PPS for company projects. The terms & conditions for academic projects have been worked on during the year but were not possible to finalize yet and this question will come up again in 2024. Also, the submission of a suggestion of prolongation for PPS, to the inventory of research infrastructure needs to the research council, was discussed and approved in the autumn. Further questions around quality control of proteins and collection of FAIR data have also been discussed.

The Key Performance Indicators (KPI) 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 2023, PPS received 197 project applications and 168 projects were completed. The projects came from 137 unique PIs and 110 PIs had completed projects. 324 protein batches were delivered, an increase with 47% since 2022. All modules had project applications and module 3.5 (plant cells) could see their first applications at the end of the year (see comments on this module above). We conclude that PPS, even with the high number of projects already in the first year, has seen an increasing demand for proteins this year from an increasing number of users and capacity- wise we are approaching our limits. New for this year was that the proportion of projects that have been made for users from their own universities (same affiliation for user and performing lab) should also be reported and was found to be 59 and 64% for applied and finished projects, respectively. The corresponding numbers for 2022 were 64 and 67%, indicating a small but positive trend towards a nationally distributed use of PPS. We can also see increased international recognition with 24 applications (vs 7 in 2022) from foreign academics and from the industry sector with 6 (vs 4 in 2022).

The list of publications for 2023 contains 35 research articles published by our users. The number of publications is lower than that reported last year for 2022 (which was 54, but mistakenly reported as 59). We are not sure about the reasons for this but speculate that it may be an effect of unproportionally many Covid-19 research publications in 2022. We are starting to 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. Several preprints containing work from PPS have appeared in 2023 and they are likely to become peer-reviewed articles in later years.

PPS had in December 2023 58 % female employees, 43 % women in the management group, 50% 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 33 % female users (unique PIs) that sent in 35 % of the project applications, and 39 % of finished projects came from women. 38 % of protein batches were delivered to women. The proportion of projects from women falls well into the span 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 application. This risk assessment is considered by us as still valid. Some mitigation measures for “Failure to follow new trends in the field/ensure that qualified personnel stay within PPS” have been anchored in the strategic plan and operational plan 2024. An event very appreciated by our staff was the startup meeting for PPS staff and a second staff retreat was hosted in 2023 by the Stockholm node and further yearly retreats are planned. To promote further career-development possibilities, like courses and relevant meetings for core facility staff, PPS worked towards a group membership in the non-profit association Core Technologies for Life Sciences (CTLS).

PPS has earlier decided to follow the 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. At this year’s PPS staff retreat, Prof. Ario DeMarco held a presentation for all PPS staff about the background and details of these guidelines. The management group has during 2023 agreed to use the following methods for quality control: 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 (if SEC is used as the last purification step, this analysis may be omitted). A UV-spectrum should also be run. So far, these analyses have not been charged extra, but discussions around this will probably be needed, as these analyses often both require more protein and are more work-intense, and it will be more costly for PPS and/or the user. A common project report template was introduced, containing all the minimal information necessary for the user to be included in future publications. Besides being sent to the user, the reports will be deposited in our database for future reference.