New BIOTECHNOLOGY 58 (2020) 45–54

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New BIOTECHNOLOGY
journal homepage: www.elsevier.com/locate/nbt

Full length Article

High throughput generation of a resource of the human secretome in

mammalian cells
Hanna Tegela, Melanie Dannemeyera, Sara Kanjea, Åsa Sivertssona,b, Anna Berlinga,
Anne-Sophie Svenssona, Andreas Hobera,b, Henric Enstedta, Anna-Luisa Volka,
Magnus Lundqvista, Mona Moradia, Delaram Afsharia, Siri Ekblada, LanLan Xua, Malin Westina,
Faranak Bidada, Lovisa Holmberg Schiavonec, Rick Daviesd, Lorenz M. Mayre,f, Sinead Knightd,
Sven O. Göpelg, Björn G. Voldborgh, Fredrik Edforsa,b, Björn Forsströma,b, Kalle von Feilitzena,b,
Martin Zwahlena,b, Johan Rockberga, Jenny Ottosson Takanena, Mathias Uhléna,b,h,
Sophia Hobera,*
a
Department of Protein Science, School of Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden
b
Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden
c
Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden
d
Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK
e
Syncona Investment Ltd, London, UK
f
ETH Zurich, B-BIOL, Molecular Health Sciences, Zurich, Switzerland
g
Bioscience Metabolism, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceutical R&D, AstraZeneca, Gothenburg, Sweden
h
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark

A R T I C LE I N FO A B S T R A C T

Keywords: The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic un-
secreted proteins derstanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a
protein production high-throughput mammalian cell factory is presented that was established to create a resource of recombinant
protein purification full-length proteins covering the majority of those annotated as ‘secreted’ in humans. The full-length DNA se-
high-throughput
quences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were
transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and
analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood
were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins
were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate
targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving
pancreatic β-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be
rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an
attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production
and purification system has been successfully established to create a unique resource of the human secretome.

Introduction the human body, knowledge of the human secreted proteins must be
developed. Such proteins can be used in a number of different assays in
A major impediment in biological sciences today is the accessibility order to explore their functions, analyze their structures and acquire
of well-validated full-length proteins to explore their characteristics and knowledge of their interaction partners. The generation of proteins on a
functions. In order to increase understanding of the inherent control of whole proteome scale requires the selection and development of unit

Abbreviations: HSP, Human Secretome Project; ECD, Extracellular domain; CHO, Chinese hamster ovary; HEK, human embryonic kidney; MS, mass spectrometry;
PDO, dissolved oxygen; FA, formic acid; FDR, false discovery rate; DDA, data-dependent acquisition; DIA, data independent acquisition; MAFA, transcription factor
MafA; SOX9, transcription factor SOX9; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor
⁎
Corresponding author.
E-mail address: [email protected] (S. Hober).

https://doi.org/10.1016/j.nbt.2020.05.002
Received 12 March 2020; Received in revised form 4 May 2020; Accepted 30 May 2020
Available online 02 June 2020
1871-6784/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
H. Tegel, et al. New BIOTECHNOLOGY 58 (2020) 45–54

operations suitable for high-throughput production, enforcing the need for the development of pharmaceuticals. The expressed proteins are
to address strategic and technical issues, such as the choice of pro- affinity purified in a high-throughput setting and thereafter analyzed
duction host, expression system and downstream purification process; regarding purity, yield and identity. Expression data in a mammalian
validation of the generated proteins also needs to be taken into account. recombinant host cell, CHO, for 2,189 gene constructs are reported
The human proteome comprises ca. 20,000 non-redundant proteins which allow analysis of the relationship between protein characteristics
(www.ensembl.org), defined as one representative isoform from every and yield. Moreover, data are reported on an alternative expression
gene locus. Of these 20,000, more than 2,500 belong to the group of system, in the human cell line HEK 293, for a selection of the clones that
proteins that are predicted to be secreted from the cell, the secretome. show degradation patterns when produced in CHO-cells. The proteins
In this paper, the human secretome is defined according to the Human produced have been used for development of proteomic analysis assays
Protein Atlas classification [1], and includes genes coding for at least based on experimental mass spectrometry (MS), including retention
one protein isoform having a signal peptide and lacking a transmem- times and fragmentation spectra for the relevant peptides. Finally, the
brane region. In addition, several proteins that according to the UniProt purified proteins have been used in a phenotypic assay to identify
database (www.uniprot.org) are annotated as secreted, despite lacking factors that affect dedifferentiation of β-cells. In conclusion, we provide
a signal peptide, are included. From these criteria, it has been estimated a knowledge resource to facilitate basic and applied research covering
that the human secretome comprises 2,641 genes, 13% of all human the proteins actively secreted in human cells, tissues and organs.
genes [2,3]. A comprehensive annotation with the aim of providing
information about the final localization of secretome proteins has re- Materials and methods
vealed that of the 2,641 secreted proteins, 932 are secreted into in-
tracellular vesicles or bound to the cell membrane. Hence, a large For more detailed methods, please see the Supplementary in-
fraction of the proteins in the secretome are predicted not to be secreted formation.
extracellularly, but are instead retained intracellularly or within the
plasma membrane [2]. Among the 1,709 proteins that are secreted
extracellularly, many are predicted to remain in the close vicinity of the Construct design
secreting cell, while 730 of these are predicted to be secreted into the
blood. The latter can be divided into two groups depending on the route Constructs were designed based on sequence information found in
of secretion. They may either be secreted through the normal cellular the UniProt and Ensembl databases. For classical secreted proteins, the
secretion pathway, released by induced vesicular secretion, or cleaved endogenous signal peptide was replaced with the CD33 signal peptide.
from the cell surface through active release. In this paper, these proteins For non-conventional secreted proteins, not having a predicted signal
are divided into two groups: the first belong to the group designated peptide, the CD33 signal peptide was added to the N-terminus after
“Blood” and the second to the group designated “Blood – other main removal of the starting methionine in the sequence. For single-pass
location”, see definitions in Table 1. Another group of proteins also transmembrane proteins, the sequence of the extracellular domain was
included in the “Blood – other main location” category consists of selected and the CD33 signal peptide was either added to, or used to
predicted secreted proteins expressed by blood cells, but also high ex- replace, any endogenous signal peptide. All constructs were equipped
pression in other tissues. To further build knowledge about the secreted with a Protein C purification tag at the C-terminus preceded by a TEV
human proteins, a resource of full-length proteins is instrumental. protease site [4], inserted to allow for cleavage of the tag. In all cases
Here, we report on the “Human Secretome Project” (HSP), a pro- any predicted C-terminal propeptide was excluded from the construct
gram in which synthetic constructs for each gene corresponding to a and for proteins with a predicted N-terminal propeptide, constructs
predicted secreted protein as well as 542 extracellular domains (ECDs) were designed both including and excluding the propeptide.
have been generated and used for protein expression in mammalian cell
factories. The ECDs are included since these are secreted by the same Plasmid preparation
machinery as the majority of the secretome. Furthermore, it is a very
interesting group of proteins, both to understand cell biology but also All designed constructs were synthesized and cloned into the ex-
pression vector pQMCF-1-MCS (Icosagen Cell Factory OÜ, Tartu,

Table 1
Summary of the definitions of the different annotation categories
Category Definition

Blood Proteins secreted into the blood

Blood - other main location Proteins secreted into the blood with their main function related to another location, for example, membrane receptors with proteolytically cleaved
secreted form or proteins secreted from blood cells having a majority of the expression in other tissues

Digestive system Proteins locally secreted to the gastrointestinal tract, for example, digestive enzymes

Male locally Proteins locally secreted in male tissues, for example, proteins related to sperm maturation

Female locally Proteins locally secreted in female tissues, for example, the oocyte zona pellucida proteins

Brain locally Proteins locally secreted in the brain, for example, some neuropeptides

Matrix Locally secreted to or in the extracellular matrix, including both structural and matricellular proteins

Other locally Proteins locally secreted by either specific tissues not included above, for example, skin, lung, eye and ear, or by a specific cell type, or by several
different tissues

Secreted - no data Secreted proteins with little data and for which the location cannot be determined

ECD The extracellular domain of single-pass transmembrane proteins

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H. Tegel, et al. New BIOTECHNOLOGY 58 (2020) 45–54

Estonia) by GeneArt (Thermo Fisher Scientific, Waltham, MA, USA). Protein purification and analysis
The plasmids were transformed into Top10 cells using standard pro-
cedures. Single colonies were chosen for cultivation and prepared using Purification of the produced proteins was performed on ÄKTAxpress
the Plasmid Plus midi Kit (Qiagen, Hilden, Germany) according to the systems (GE Healthcare) with affinity chromatography using a Protein
manufacturer’s instructions. C-tag antibody matrix [5,6] packed in 1 ml HiTrap columns, followed
by buffer exchange using 2 x 5 ml HiTrap desalting columns (GE
Production of secreted proteins in CHO cells Healthcare) at standard flow rates. Buffers used were wash buffer (20
mM Tris, 150 mM NaCl, 2 mM CaCl2, pH 7.5) and elution buffer (20
All secreted proteins from CHO cells were produced by using a mM Tris, 100 mM NaCl, 5 mM EDTA, pH 7.5). After filtration of the
transient expression system from Icosagen Cell Factory; the QMCF supernatant using a 0.45 μm syringe filter and addition of CaCl2 to 2
Technology (Icosagen Cell Factory OÜ). mM, the samples were loaded onto the column and unbound proteins
were washed out. Elution was performed with elution buffer and col-
Small-scale lected in the loops of the ÄKTAxpress system before buffer exchange to
The cells were pelleted and resuspended in medium containing 1x PBS (2 mM NaH2PO4, 8 mM Na2HPO4, and 150 mM NaCl, pH 7.4).
plasmid DNA and transfected using a BTX electroporator (twin wave Proteins produced at medium-scale were purified similarly but with
HT96 well system gemini X2, Harvard apparatus, Holliston, MA, USA). larger affinity columns (2 x 5 ml per 200 ml supernatant), a HiPrep 26/
After transfection the cells were added to fresh pre-warmed medium 10 desalting column and adjusted flow rates.
containing penicillin-streptomycin and grown in fed batch for 13 d. 48 Protein concentrations were determined using absorbance at 280
h after transfection the cells were diluted and after six days protein nm and analysis of protein purity and identity were performed by SDS-
production was promoted by adding feed and shifting the temperature PAGE analysis and WB including deglycosylation of the proteins using
from 37 to 30 °C. A second feed was added 9 d after transfection. At day Mix II (NEB, Ipswich, MA, USA) according to manufacturer’s instruc-
13 the supernatant was clarified by centrifugation and serine-protease tions.
inhibitor was added before sample storage at −20 °C.
Production of standards for targeted proteomics analysis (QPrESTs and
Medium-scale QTag)
Cells were transfected by chemical transfection using reagent 007
(Icosagen Cell Factory OÜ). After 20-24 h, pre-warmed medium was The production of both HisABPOneStrep (QTag) and stable isotope-
added. Protein production was promoted after 72 h by adding 10% labeled protein fragments (QPrESTs) used for protein quantification
Basic Feed (Xell AG) and a temperature decrease to 30 °C. Feeding was was essentially done as described in [7]. Absolute quantification of the
continued at day 6, day 8 and day 10. At day 13 the supernatant was QTag was obtained by amino acid analysis. The QPrESTs were pro-
clarified by centrifugation and stored as described above. duced in an auxotrophic E. coli strain according to [8]. The expressed
QPrESTs were purified by the standard workflow used within the
Pilot-scale Human Protein Atlas for PrEST production [9].
Cells were transfected as described in the small-scale section. The
transfectants were selected after at least 14 d by diluting the cells every Preparation of samples for protein identification using mass spectrometry
second day with Geneticin (Gibco, Thermo Fisher Scientific) starting at (MS)
48 h. The culture volumes were simultaneously increased to the desired
start volume for WAVE, Cellbag cultivation. Selected cells were trans- The secretome protein products were diluted to a final concentra-
ferred to 20 L Cellbags (GE healthcare) containing pre-warmed selec- tion of 1.5 μM in 50 mM ammonium bicarbonate into a 96-well plate.
tion medium. pH and dissolved oxygen (PDO) were monitored 15-30 pmol of each protein was transferred to a new 96-well plate and
throughout the production process. Cell selection continued for 3-4 d in mixed with the corresponding isotope-labeled standard and quantifi-
Cellbags until final production volume was reached. To increase protein cation tag. The samples were reduced (5 mM DTT, 30 min at 56 °C), and
production, the temperature was shifted to 30 °C while proceeding with alkylated (10 mM 2-chloroacetamide/2-iodoacetamide, 30 min in the
feeding on a 2-day basis. Harvest was performed after 6-8 d in pro- dark at RT) and cleaved with 200 ng of proteomics grade porcine
duction phase using 5 and 0.2 μm filters and stored as described the in trypsin (Sigma-Aldrich) overnight at 37 °C and thereafter quenched by
small-scale section. addition of formic acid (FA) to a final concentration of 1%. After di-
gestion the samples were vacuum dried and stored at −20 °C.
Production of secreted proteins in HEK 293 cells in small-scale
Analysis using data-dependent acquisition mass spectrometry
For transient production Expi293 F cells (Thermo Fisher Scientific)
were used. Plasmid DNA was diluted in expression medium and added The samples were analyzed using one of two different liquid chro-
to 1 ml PEI MAX (Polysciences, Inc, Warrington, PA, USA) (1 mg/ml) matography (LC)-system setups. The first setup used a Dionex Ultimate
and incubated for 15 min before addition to the cells. 24 h after 3000 (Thermo Fisher Scientific) equipped with a trap column and a 25
transfection, cultures were diluted with expression medium. Harvesting cm analytical C-18 column (Thermo Fisher Scientific). For the mobile
was performed by centrifugation 4 d after transfection. The cultures phase, solvent A (3% acetonitrile (ACN), 97% H2O, 0.1% FA) and sol-
were stored as described above. vent B (95% ACN, 5% H2O, 0.1% FA) were used. Peptides corre-
sponding to 1 pmol per protein were separated using a gradient of 4-
Analysis of protein production 40% solvent B over 9 min, 0.5 μl/min in solvent A. The second setup
used a Dionex Ultimate 3000 equipped with a 15 cm analytical C18-
The CHO cell cultures were assessed for protein secretion (“first column (Thermo Fisher Scientific). Peptides corresponding to 4 pmol
analysis”) 6 d post-transfection (3 d for medium-scale) using Western per protein were separated using a gradient of 5-37% solvent B over 8
Blot (WB) analysis and at harvest using WB and sodium dodecyl sulfate min, 150 μl/min. Both separation setups were connected to a Bruker
– polyacrylamide gel electrophoresis (SDS-PAGE). The HEK cell cul- Impact II (Bruker Daltonics, Billerica, MA, USA) and the samples were
tures were analyzed at harvest using WB and SDS-PAGE. In WB a pri- analyzed in data-dependent acquisition mode, with a 3 s cycle time. The
mary rabbit antibody against the C-tag, GTX18591 (GeneTex, Irvine, method performed a survey scan from 150 to 2,200 m/z (1 Hz) fol-
CA, USA) was used for protein detection. lowed by MS/MS scans acquired dynamically (2,500 cts =8 Hz to

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25,000 cts =32 Hz). The dynamic MS/MS acquisition selected ions at 20X using BP filter 445/50 for Hoechst and BP filter 525/50 for
with a charge state of 2-5 and implemented a smart exclusion (5x) set to MAFA and SOX9. Image segmentation and analysis were performed
30 s. using Columbus™ software (PerkinElmer, Waltham, MA, USA).

Data analysis of data-dependent acquisition results Single-Cell RNA-Seq of Pancreatic Islets

The raw data obtained were searched using MaxQuant (version In brief, human tissue and primary islets were purchased from
1.5.7.0) [10] to confirm the identity of the proteins. MS/MS spectra Prodo Laboratories Inc. (Irvine, CA, USA). The use and storage of
were searched in batches against a database containing all proteins human islets and tissue samples were performed in compliance with the
produced in the same cultivation batch using Andromeda [11] with the Declaration of Helsinki, ICH/Good Clinical Practice and was approved
entire CHO proteome as background (UP000001075, retrieved by the independent Regional Ethics Committee. Human islet samples
2018.03.27, 23,888 entries) and a list of the most common con- (85%–95% pure) were cultured for 4 days in complete Prodo Islet
taminants. The multiplicity was set to 2, allowing for quantification Media Standard to recover after arrival. Islets were dissociated and
against the stable isotope-labeled standard, with Arg10 and Lys8 se- distributed by Fluorescence Activated Cell Sorting into 384-well plates.
lected as heavy labels. The false discovery rate (FDR) was set to 1% Single-cell RNA-seq libraries were produced with the Smart-seq2 pro-
both at peptide and protein level and the minimum peptide length was tocol [15]. Sequencing was carried out on an Illumina HiSeq 2000
set to 4. generating 43 bp single-end reads. Sequence reads were aligned toward
the human genome (hg19 assembly) using STAR (v2.3.0e), and un-
Data independent acquisition (DIA) library generation iquely aligned reads within RefSeq gene annotations were used to
quantify gene expression as RPKMs using rpkmforgenes[16]. For a
Peptides from the previously prepared MS samples were pooled in more detailed description see [15].
sets of eight proteins and analyzed using the same LC-MS/MS setup as
for the analysis of protein products. Peptides were loaded onto a trap Results and Discussion
column, washed for 5 min with 100% of solvent A, separated on a 25
cm analytical C18 Easy-Spray column gradient of 6-28% solvent B and A protein factory for production of the human secretome
analyzed using a Top5 data-dependent acquisition (DDA) method. Raw
files were searched in MaxQuant version 1.5.2.8 against the secretome For production of the human secreted proteins and selected ECDs of
protein product sequences and the MS/MS files from the MaxQuant single pass transmembrane proteins, a standardized protein production
searches were used to build a spectral library in Skyline [22]. All pro- pipeline was set up (Fig. 1A). The system was based on a mammalian
tein sequences can be found in the Panorama repository (https:// CHO cell host system in combination with semi-stable transfection of
panoramaweb.org/human_secretome.url, username: panorama + clones generated by gene synthesis. To enable production and sub-
[email protected]). sequent purification of all proteins, the recombinant proteins were
synthesized with an N-terminal signal peptide (CD33) and a C-terminal
DIA analysis of pooled plasma samples purification handle (Fig. 1B). After transfection of the expression vec-
tors the proteins were transiently produced in mammalian CHO cells
Two different pools of human plasma samples obtained from using the QMCF Technology [17] and subsequently purified using an
healthy donors were digested as described in [7]. The analysis was antibody-based chromatography resin with a calcium ion-dependent
performed on the same instrument setup and LC-gradient as described affinity for the Protein C-tag, included at the C-terminus of the re-
above for DIA library generation, but two of the samples were analyzed combinant protein. This tag enables mild elution by the use of a che-
using a 50 cm C18 EASY-Spray column (Thermo Fisher Scientific) and lating elution buffer [18]. The purity and protein identity of the various
the MS was operated in a DIA mode. For all samples a total of 1 μg of target proteins were analyzed by SDS-PAGE, WB and MS/MS (Fig. 1A).
peptides was injected onto the column. One sample was also injected 12 Initially, protein production was performed in a small-scale setting
times and analyzed in DIA using small isolation windows, and with the with a final culture volume of 50 ml (Fig. 1C). The recombinant pro-
multiple injections covering the same range as the other DIA methods. teins were purified from the conditioned medium with a protocol using
Raw data files were imported into Skyline and matched against the the C-terminal purification tag. For the proteins produced at small
curated spectral library. Peaks with matching fragmentation spectra scale, 1 ml of affinity matrix was used for each culture. The mean
were integrated and MS2 peak intensities were extracted for peptides protein amount achieved in small scale was 755 μg for all proteins
that could be detected in the plasma samples. Extracted ion chroma- successfully produced (Fig. 2B). A maximum amount of 5 mg pure
tograms were uploaded to Panorama, access as above. protein was achieved for C-X-C motif chemokine 5 (CXCL5) from a
single 50 ml culture. However, depending on the intended application,
EndoC-βH1 Dedifferentiation screening assay larger protein amounts might also be needed. Therefore, a medium-
scale protocol was developed (Fig. 1D), for which the final culture
EndoC-βH1 cells (Univercell Biosolutions, Toulouse, France) were volume was 900 ml. This large volume of conditioned medium was
cultured according to [12]. The cells were dispensed at a density of 6 × purified using 40 ml of affinity matrix and normally generated over 10
103/well and incubated under standard culture conditions for 24 h. mg of pure protein with a maximum, so far, of 153 mg. The mean
Cells were then treated with fibroblast growth factor FGF2 and neutral protein amount for all proteins successfully produced at medium scale
control (media) according to [13] or with the secretome library ac- was 35 mg (Fig. 5A). Finally, to be able to produce even larger amounts
cording to [14]. The secretome library applied consisted of 812 protein of proteins, a protocol for a WAVE Bioreactor system with 20 L Cellbags
samples (corresponding to 765 unique genes) in 3-point concentration and a final culture volume set to 10 L was developed (Fig. 1E). With this
response. Dosed cells were incubated for another 96 h before fixation, setup, a stable pool was generated prior to protein production and up to
permeabilization and labelling with MAF BZIP Transcription factor A 1 g of purified protein could be produced.
(MAFA) (catalog no. #79737 Cell Signaling Technology, Inc., Danvers,
MA, USA) and SRY-Box Transcription factor (SOX9) (catalog no. Small-scale production
ab196184 Abcam PLC, Cambridge, UK) antibodies. Confirmation
screening was carried out in a 10-point concentration response. All By using the small-scale protocol (Fig. 1C), 2,189 different genes
images were acquired using CV7000 (Yokogawa) confocal microscopy have been initiated in the production pipeline using CHO cells as the

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H. Tegel, et al. New BIOTECHNOLOGY 58 (2020) 45–54

Fig. 1. A summary of the different production pipelines is shown. A) An outline of the protein production pipeline including production, purification and analyses.
The first step in the standardized high-throughput protein production pipeline used in the HSP is construct design. The constructs are then synthesized and cloned
into the expression plasmid. All plasmids are prepared and sequence verified before protein production. The Protein C-tagged target proteins are then purified using
an automated affinity purification setup. Purified proteins are identified and quantified with MS/MS and purity and glycosylation patterns are determined using SDS-
PAGE and WB. B) All proteins produced in the HSP are produced with an N-terminal signal peptide, CD33, and a C-terminal purification handle based on the Protein
C-tag purification tag. Between the purification tag and the protein, a TEV protease site was inserted to allow for cleavage of the tag, if needed. The CMV promoter is
used to control the protein production. C–E) shows the production protocol for the three different scales used, small- medium- and pilot-scale respectively.

production host (Supplementary Table S1). From this high-throughput The protein yields after production and purification varied among
production system without optimization for individual proteins 1,276 the proteins (Fig. 2B). Many of the secreted proteins are post-transla-
different proteins (58%) were successfully produced and purified. The tionally modified during translocation through the secretory pathway.
success rate for each annotated category, see definitions in Table 1, is In order to assess the size and purity of the produced proteins and also
shown in Fig. 2A and interestingly the highest success rate was to achieve information on the degree of glycosylation, a mixed degly-
achieved when producing proteins that are naturally secreted to the cosylation enzyme kit was used. This is exemplified in Fig. 2C showing
digestive system and to blood, 78% and 66%, respectively. Also, the heterogeneous and oversized bands for two of the proteins after CHO
selected extracellular domains (ECDs) showed a high success rate production and single bands of expected size after deglycosylation. As
(71%). Proteins with the lowest success rate regarding production and expected, the CHO host system often yields proteins with differential
purification are annotated as being secreted to the cell matrix (33%). glycosylation patterns, a phenomenon that has been reported pre-
Furthermore, the group of proteins that is less understood and com- viously [19].
prises proteins that remain to be explored is also among those with very
low success rate (37%).

Fig. 2. Bioproduction of the human secretome in CHO cells in small-scale cultivations. A) The success rates for the CHO clones are shown. The overall success rate
was almost 60%, although differing considerably between different groups based on predicted localization (Table 1), where proteins targeted for the digestive system
are the group with the highest success. B) The amount of the different target proteins generated by the stream-lined approach; see Suppl. Table 2 for details regarding
protein amounts. C) SDS-PAGE gels showing three representative proteins after purification: the first lane shows the molecular marker, the second lane shows
purified protein sample and the third lane the same protein after deglycosylation. The enzymes from the deglycosylation kit are indicated by their respective names to
the right.

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H. Tegel, et al. New BIOTECHNOLOGY 58 (2020) 45–54

Quality control of the produced and purified proteins in the cell matrix were overrepresented. Among the proteins annotated
as being secreted locally in the brain, proteins failed due to production
During the production process the quantity and biophysical quality problems were slightly overrepresented, although this group only in-
of the target proteins produced were analyzed at different time points. cludes 33 proteins. To understand if hydrophobicity could have had an
The first analysis of the proteins from small- and medium-scale pro- impact on success rate, the hydropathy of the amino acid sequences of
duction took place 6 and 3 days after transfection, respectively. all target proteins was analyzed using the Kyte and Doolittle hydro-
Supernatant for which the WB analysis of the culture media showed the pathy scale [20]. In all statistical analyses the group “fail others” (48
expected protein band were regarded as having passed the analysis proteins) was disregarded, as it includes proteins that failed for a
while proteins with a blank WB required further evaluation by analysis number of different reasons, among them diverse technical failures.
also of the cell lysate on WB. Where the WB was blank for conditioned These data were used to understand if there was any identifiable dif-
medium as well as lysate, the protein was failed and classified as “no ference in the distribution of hydrophobic proteins among the different
production”. Proteins that showed a band in the lysate lane but not in fail groups and the successfully produced proteins. Although the pro-
the lane for conditioned medium were clearly produced but not se- teins in the data set are rather hydrophilic, there is a significant dif-
creted and therefore failed and classified as “no secretion”. For the ference in hydrophobicity between proteins successfully produced and
proteins that passed the first analysis, production continued until har- those failed due to degradation, where the latter are slightly less hy-
vest at day 13. At harvest, an aliquot of the supernatant was analyzed drophobic (Supplementary Fig. S1). Also, when analyzing the re-
on SDS-PAGE as well as WB. Proteins with a clear band of expected size lationship between length and production success, a significant differ-
proceeded to purification while proteins with a weak target band or ence was observed between successfully produced proteins and those
showing < 80% of expected size were failed and classified as “low failed due to degradation with larger proteins being clearly over-
production” and “degradation”, respectively. Purity was confirmed using represented in the fail category (Supplementary Fig. S2A). This may
SDS-PAGE as well as WB including analysis of deglycosylated protein explain why the group of proteins that are secreted to the cell matrix,
samples. The identity of the proteins that fulfilled the purity criterion of which include many large proteins such as collagens and laminins, had
at least 80% of the sample having the expected size, was finally verified a lower success rate compared to the other groups. (Fig. 3, Supple-
using MS/MS. mentary Fig. S2B).

Using human cell line HEK 293 to rescue difficult proteins

Reason for failure in the small-scale production pipeline
For proteins that failed production in CHO cells, the possibility that
To understand if there was any common feature among the proteins these could be rescued by expression in a human cell line was in-
that were difficult to produce, the possible connection was investigated vestigated. Some of the constructs previously failed due to degradation
between failure rate, failure reason and protein characteristics of size were therefore transfected into HEK 293 cells and transiently produced
and hydrophobicity, as well as expected localization. All the proteins in a 4-day cultivation with a final culture volume of 40 ml. By using this
that were produced and purified passed through different quality as- protocol, up to 2.5 mg of pure protein was generated from a single
surance steps where they were sorted according to the analytic results culture. Many proteins that showed degradation in CHO cells were
(see above), i.e. passed or failed. Within the group of failed proteins successfully produced in the human cell line, see examples in Fig. 4
there were five different classes according to the reason for failure, (Supplementary Table S2). Out of 126 protein constructs that had
namely: degradation (218), low production (254), no production (134), earlier failed in the CHO cell factory due to degradation, 86 (68%) were
no secretion (183) and a small group with inconsistent data, denoted successfully rescued by changing the production host from CHO to HEK
‘others’ (48). Regardless of annotated localization, all groups had pro- 293. These results suggest that using a human cell line would be an
teins in all five fail-classes (Fig. 3). However, among the group of attractive option for difficult to produce proteins in the standard bio-
proteins that failed due to degradation, those annotated with a function manufacturing CHO host cell line due to degradation. One possible
explanation for why these proteins do not degrade in the human cell
line is the shorter production protocol used. Furthermore, hosts of
different origin with differences in protein processing machinery, may
have different impacts on the degradation pattern.

Medium-scale production

To be able to meet the demand for larger protein amounts than were
possible to generate in the small-scale production pipeline, a medium-
scale pipeline was developed (Fig. 1D). All constructs selected for
production at medium-scale had previously been successfully produced
at a small-scale and to date 369 different proteins have been produced
with an average yield of > 35 mg purified protein (Fig. 5A). For 86% of
the proteins, > 10 mg purified product was achieved, with the highest
amount being 152 mg achieved for proprotein convertase subtilisin/
kexin type 9, PCSK9, an important target protein in immunotherapy for
atherosclerosis [21]. When comparing the amount of pure protein from
the different scales of production, it could be concluded that the
medium-scale protocol generally performed better than the small-scale
(Fig. 5B) without lowering the quality of the end product (Fig. 5C).

Fig. 3. Reasons for failure when producing the secretome in small scale. The Pilot-scale production
bar plot shows the different causes of failure in the groups of final predicted
biological destination for the 837 out of 895 failed proteins in small scale To be able to increase further the amount of protein produced, a fed-
protein production for which a final reason for failure have been determined. batch protocol using WAVE Bioreactor systems was developed, aiming

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Fig. 4. Rescue of difficult proteins in human cell line HEK 293. A) Examples of WBs with samples from the harvest in CHO (left WB) and HEK (right WB).

for a final culture volume of 10 L (Fig. 1E). This protocol has to date serial quantification. First, the light peptides from the quantification tag
been used for production of six different proteins, generating amounts are compared to the heavy labeled peptides from the QPrEST and
of up to 1 g pure protein for antithrombin (SERPINC1). When com- thereafter the heavy labeled peptides from the QPrEST are compared
paring the amount of pure protein per volume cultivation from the with the corresponding light peptides from the produced protein.
three different scales, it is apparent that the protein production levels Thereby, both the amount of QPrEST and produced protein can be
were highest with pilot-scale (Supplementary Table S3). Even though determined in a single MS experiment. A comparison between the tar-
the number of proteins was rather small, their quality was similar to geted proteomics analysis and the determination of protein concentra-
that achieved on a smaller scale (Fig. 6), and thus increasing the scale tions by spectrophotometry demonstrated that the latter more often
did not compromise the purity of the final product as assessed by SDS- generated over- or under-estimation of the concentration of the target
page, WB and deglycosylation. protein (Fig. 7, Supplementary Table S4). Hence, it was decided to
determine the absolute concentration of each protein using the MS
Development of an MS-based quantification method based workflow and the production levels for each construct is pre-
sented in Supplementary Table S4.
To simultaneously determine concentration and identity of the
produced proteins, an MS-based method for duplex serial absolute Use of the secretome resource to develop proteomics assays
quantification was developed. This method is based on spike-in of
stable isotope-labeled protein fragments (QPrESTs) corresponding to Data-independent acquisition (DIA) mass spectrometry can provide
the target proteins [7]. The isotope-labeled QPrESTs are fused to a highly quantitative MS2-data for thousands of proteins in a single
QTag, which is used for purification and subsequent quantification. analysis, but requires libraries of peptide fragmentation spectra and
Heavy labeled peptides coming from the QPrESTs are, together with a retention times for identification of peptide peaks during the data
light quantification tag (QTag) of known concentration, used to enable analysis step [22,23]. These fragmentation spectra are often based on

Fig. 5. Bioproduction of the human secretome in CHO cells in medium-scale cultivations. A) The amount of the different target proteins generated by the stream-
lined approach, see Suppl. Table 3 for details regarding expression levels. B) Comparison of the amount protein achieved in small scale production with the amount
achieved at medium-scale. It is evident that in most cases the larger scale produces more per unit cultivation volume. C) SDS-PAGE gels showing the same three
proteins after purification as for the small scale (Fig. 2C): the first lane shows the molecular marker, the second lane shows the purified protein and the third the
purified protein after a deglycosylation treatment.

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Fig. 6. Bioproduction of the human secretome in CHO cells using WAVE

bioreactors. SDS-PAGE gels showing the three proteins earlier produced in
small and medium-scale (Figs. 2C and 5 C) after production and purification in
pilot-scale, the first lane showing the molecular marker, the second lane
showing the purified protein and the third lane the purified protein after a
deglycosylation treatment. As can be seen, the quality of the proteins is similar
regardless of the scale that had been used for the production step.

shotgun proteomics analysis of complex samples which might result in

low quality spectra where low abundant proteins are often missing. Fig. 8. Use of the secretome resource to set up a DIA analysis. A) Schematic
Therefore, it was decided to make use of the secretome resource to view of the proteomics assay development. Proteins were combined into pools
establish a high-quality spectral library by analyzing trypsin digested of eight and digested with trypsin before analysis in shotgun-MS mode. Peaks of
equimolar pools of the purified proteins in shotgun mode (Fig. 8A). In detected peptides were inspected manually and their fragmentation spectra and
total, secreted proteins corresponding to 368 unique genes were retention times were used for data extraction through DIA. B) Beeswarm plot
screened and the fragmentation spectra were manually verified using showing the secretome proteins and ECDs for which DIA assays could be es-
the Skyline software [24]. For 340 of the proteins, DIA assays could be tablished and whether they could be detected (red) or not (black) in human
plasma analyzed through DIA. The proteins are divided into their respective
created based on at least one tryptic peptide, while 28 of the proteins
secretome classification categories and the y-axis shows the maximum nor-
did not result in any peptide fragmentation spectra. Human plasma
malized mRNA expression (NX) in human normal tissue (according to http://
samples were then analyzed and peptide data corresponding to 83 www.proteinatlas.org).
proteins could be extracted using the developed spectral library, while
257 proteins could not be detected using this DIA proteomics analysis
(Fig. 8B). As expected, proteins that are actively secreted into the blood
were detected in plasma to a larger extent than any other category,

Fig. 7. Absolute quantification by MS of the produced proteins. By utilizing QPrESTs originating from the Human Protein Atlas[1] absolute quantifications of the
produced proteins were made using LC-MS/MS. A). The histogram shows the difference between the absolute concentration determined by LC-MS/MS and the
concentration obtained through absorbance measurements and B) the scatterplot, shows the ratio between spiked in QPrEST and protein product used for the
quantification.

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differentiation status in the human pancreatic β-cell line EndoC-βH1,

which has recently been shown to reflect the major features of primary
human β-cell dedifferentiation [13,26]. A subset of the proteins pro-
duced comprising 765 unique protein genes (Supplementary Table S5)
was further applied to explore the secretome resource for stimuli-in-
duced changes in phenotypic screens, using physiologically relevant
cells to aid in the development of drug candidates as described pre-
viously [14,27–30]. The library of proteins generated in-house was used
to explore the induction of dedifferentiation of the EndoC-βH1 cell line.
Dedifferentiation was monitored by measuring changes in the sub-
cellular location and expression of the β-cell marker MAFA (a key
regulator of glucose stimulated-insulin secretion) and the pre-endocrine
marker SOX9 (Fig. 9A). Interestingly, FGF9 showed a higher activity
than the FGF2 positive control, but FGF1, FGF4 and FGF18 also affected
differentiation state (Fig. 9B). As far as we are aware, the activity of
several of these fibroblast growth factors has not been described before
in this context. Single cell analysis of FGFR expression on primary
human islets showed that FGFR1 was dominantly expressed (Fig. 9C).
Taken together, the data suggest that therapeutic intervention could be
achieved by inhibition of FGFR1 signaling with no additional signaling
pathways identified that maintain the dedifferentiated state of β cells.

Conclusion

The human secretome is a highly interesting group of proteins both

in studies of human biology and as targets for the development of new
drugs and diagnostics. Here, we report a high-throughput mammalian
cell factory for recombinant expression of the secretome in CHO and
HEK 293 cells. 1,276 human proteins were produced, successfully
purified and analyzed regarding concentration and purity. All the data
from the mammalian cell factories are available to enable further ex-
plorations of factors important for successful bioproduction in CHO
and/or human HEK 293 cell lines. This protein resource has been used
both for generating a spectral library used to identify proteins in MS-
based data independent acquisition (DIA) workflows aimed at analysis
of human blood and for phenotypic assays involving β-cell dediffer-
Fig. 9. Use of the secretome resource in phenotypic assays. A) Schematic view entiation. The phenotypic assay generated several actives from the fi-
of the β-cell dedifferentiation assay. A subset of the secretome library consisting
broblast growth factor family, and the proteomics assays were used to
of 765 unique protein genes was screened on the human β-cell-like EndoC-βH1
analyze the presence or absence of the target protein in human plasma.
cell line to identify secreted proteins that affect the differentiation state of the
cells [13]. Transcription factors SOX9 and MAFA were used as markers of
dedifferentiation. EndoC-βH1 cells were treated with secretome proteins at Acknowledgments
three concentrations. As positive control FGF2 was used and for the baseline,
neutral cell media. B) All data were normalised to neutral and FGF2 positive Funding was provided from the Novo Nordisk Foundation, the Knut
control (MAFA inhibitory and SOX9 stimulatory control). Confirmatory 10 and Alice Wallenberg Foundation, AstraZeneca and Vinnova (2017-
point concentration response studies confirmed FGF9, FGF4, FGF18 and FGF1 02105). We acknowledge the entire staff of the Human Protein Atlas
as inducers of EndoC-βH1 dedifferentiation with FGF9 showing a greater in- program for their valuable contributions.
crease in SOX9 and decrease in MAFA than the FGF2 positive control (indicated
by *). C) mRNA analysis of the FGF receptor expression in primary human islets Appendix A. Supplementary data
showed that predominantly FGFR1 is expressed.

Supplementary material related to this article can be found, in the

although some belonging to the non-blood categories, especially those online version, at doi:https://doi.org/10.1016/j.nbt.2020.05.002.
with high relative mRNA expression levels in at least one tissue, were
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