Bioactive VEGF-C from E. coli

Rauniyar, Khushbu; Akhondzadeh, Soheila; Gąciarz, Anna; Künnapuu, Jaana; Jeltsch, Michael

doi:10.1038/s41598-022-22960-0

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Published: 28 October 2022

Bioactive VEGF-C from E. coli

Khushbu Rauniyar¹^na1,
Soheila Akhondzadeh¹^na1,
Anna Gąciarz²,
Jaana Künnapuu¹ &
…
Michael Jeltsch^1,2,3

Scientific Reports volume 12, Article number: 18157 (2022) Cite this article

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Abstract

Vascular endothelial growth factor-C (VEGF-C) stimulates lymphatic vessel growth in transgenic models, via viral gene delivery, and as a recombinant protein. Expressing eukaryotic proteins like VEGF-C in bacterial cells has limitations, as these cells lack specific posttranslational modifications and provisions for disulfide bond formation. However, given the cost and time savings associated with bacterial expression systems, there is considerable value in expressing VEGF-C using bacterial cells. We identified two approaches that result in biologically active Escherichia coli-derived VEGF-C. Expectedly, VEGF-C expressed from a truncated cDNA became bioactive after in vitro folding from inclusion bodies. Given that VEGF-C is one of the cysteine-richest growth factors in humans, it was unclear whether known methods to facilitate correct cysteine bond formation allow for the direct expression of bioactive VEGF-C in the cytoplasm. By fusing VEGF-C to maltose-binding protein and expressing these fusions in the redox-modified cytoplasm of the Origami (DE3) strain, we could recover biological activity for deletion mutants lacking the propeptides of VEGF-C. This is the first report of a bioactive VEGF growth factor obtained from E. coli cells circumventing in-vitro folding.

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Introduction

Vascular endothelial growth factor VEGF-C is essential for the embryonic development of the lymphatic vasculature and its maintenance in the adult organism¹. Unlike VEGF-A, which is secreted as an active growth factor, the VEGF-C polypeptide (unprocessed VEGF-C) requires two proteolytic cleavages for activation². The first C-terminal cleavage (resulting in pro-VEGF-C) occurs constitutively in the oxidizing environment of the endoplasmic reticulum/Golgi apparatus and is mediated by protein convertases³. The second cleavage step takes place in the extracellular environment and is mediated by multiple enzymes, including plasmin⁴, ADAMTS3⁵, PSA/KLK3, cathepsin D⁶, and thrombin⁷. It results in N-terminally slightly different mature forms of VEGF-C which differ in their activity towards their receptors VEGFR-2 and VEGFR-3⁶. With about 9% of its amino acid residues being cysteine residues, VEGF-C belongs to the 50 cysteine-richest long (> 400 aa) proteins coded by the human genome (https://github.com/mjeltsch/cysteine). With the increasing number of cysteines, the difficulty of correct disulfide bond formation increases exponentially. Moreover, the high number of genes in most animal genomes for enzymes catalyzing disulfide bond formation and isomerization supports the idea, that VEGF-C might be difficult to produce in a prokaryotic expression host like E. coli. In addition, it is known that eukaryotic chaperones are known to aid in the correct folding of the closely related VEGF-A^8,9.

To produce VEGF-A, PlGF, and VEGF-B for crystallization purposes, solubilization from inclusion bodies and subsequent refolding under the appropriate conditions have been deployed^{10,11,12,13,14,15}. However, the refolding efficacies were never explicitly reported. Final yields of active protein have been reported to be in the range of 2–4 mg/L of bacterial culture¹⁵ or 10 g bacterial wet weight¹⁰, which translates to estimated refolding efficiencies below 1%. Contrary to the above proteins, all reported structures for VEGF-C and VEGF-D were solved using proteins from eukaryotic expression systems^16,17.

There are two principal methods to promote the correct folding of cystine-containing proteins in the cytoplasm of E. coli: (1) by modulating the intracellular redox environment¹⁸ as done in E. coli strains such as Origami, AD494 (both Novagen), or SHuffle (New England Biolabs), and (2) by co-expressing enzymes catalyzing disulfide bond formation and isomerization (as e.g. done in the CyDisCo system^19,20), reviewed by de Marco²¹. Alternatively, the oxidizing environment of the periplasm²² or extracellular space²³ can be utilized. Also, combinations of these strategies have been used²⁴. In this study, we tested four different approaches: (1) CyDisCo, (2) periplasmic targeting, (3) expression in a redox-modulated cytoplasm, and (4) in vitro folding of solubilized inclusion bodies. The latter two approaches were successful. Expression in a redox-modulated cytoplasm was first efficiently achieved by MBP-tagging VEGF-C, but we also were able to recover low levels of biological activity from non-MBP-tagged VEGF-C.

Results

The cysteine-rich C-terminal domain of VEGF-C appears to be responsible for the slow secretion of VEGF-C

If correct disulfide bond formation represented a bottleneck in the production of VEGF-C, one would expect that deleting the cysteine-rich C-terminal domain accelerates the folding and thus the biosynthesis and secretion of VEGF-C. We found that deleting the C-terminal domain (ΔC) as well as deleting both the N-terminal propeptide and the C-terminal domain (ΔNΔC) speeded up the secretion of VEGF-C. In a pulse-chase experiment, the maximal levels of VEGF-C in the supernatant were measured for the wild-type VEGF-C at 2 h, the ΔC mutant between 15 and 45 min, and the ΔNΔC mutant at 45 min (Fig. 1).

Partial lack of N-linked glycosylation does not preclude VEGF-C binding to VEGFR-3

A major limitation of bacterial hosts for the expression of secreted eukaryotic proteins is the lack of glycosylation. Because the VEGF homology domain (VHD) of VEGF-C features two N-linked glycosylation sites at amino acids 175 and 205, we investigated whether the glycosylation of VEGF-C is a requirement for receptor binding. Hence, we produced single and double glycosylation mutants of VEGF-C by replacing one or both Asn residues with Gln and Trp, respectively, resulting in six different glycosylation mutants designated N₁₇₅Q, N₂₀₅Q, and N_175,205Q, and N₁₇₅W, N₂₀₅W, and N_175,205W. Replacement by glutamine was chosen to maintain the chemical nature of the asparagine residue and tryptophan to mimic the bulky carbohydrate moiety. When expressed in 293T cells, all single mutants were able to bind VEGFR-2 and VEGFR-3, indicating that no specific interaction of any single carbohydrate chains is needed for receptor binding. However, the complete absence of glycosylation resulted in failure to be expressed (supplemental Figure S1) and secreted (Fig. 2a, b, supplemental Figure S1), similar to other secreted proteins²⁵.

VEGF-C expressed in the cytoplasm of E. coli BL21 (DE3) is found exclusively in inclusion bodies

Based on the glycosylation data, we reasoned that glycosylation would not be a per-se requirement for receptor binding or activation and we proceeded to produce VEGF-C in E. coli. We designed three different truncated cDNAs devoid of the C-terminal and N-terminal domains. These forms corresponded to the major and minor mature forms of VEGF-C as produced by 293 cells when transfected with full-length VEGF-C cDNA², and to a minimal form with the same N-terminus as the major mature form of VEGF-C, but which lacked the C-terminal sequences required for neuropilin-2 interaction (Fig. 2c, d)²⁶. When these three forms were produced in the cytoplasm of E. coli BL21 (DE3), all of the protein did aggregate in inclusion bodies (Fig. 2c), and no biological activity could be seen in the Ba/F3-VEGFR-3/EpoR assay in the soluble fraction (data not shown). Based on this observation and the suspected difficulty of disulfide bond formation, we decided to apply CyDisCo, which is a system enhancing soluble expression of disulfide-bonded proteins in the cytoplasm of E. coli.

Coexpression with a sulfhydryl oxidase and disulfide bond isomerase (CyDisCo system) does not yield active VEGF-C

Previously, we had unsuccessfully tried to express active VEGF-C in the cytoplasm of E. coli strain AD494 (DE3), which has a mutation in the thioredoxin reductase B gene, which is one of the key players in the pathways reducing disulfide bonds in the cytoplasm of E. coli²⁷ (data not shown). The more recently developed, enhanced intracellular E. coli expression system CyDisCo differs from AD494 and similar E. coli strains in that it does not modify the cytoplasmic redox environment, but only facilitates correct disulfide bonding by co-expressing enzymes catalyzing disulfide bond formation (a sulfhydryl oxidase such as Erv1p) and isomerization (human PDI)^19,20,28. CyDisCo system proved efficient for soluble expression of a variety of disulfide-bonded proteins including antibody fragments²⁸, BPTI, vtPA, resistin, Ero1α, CFS3, BMP4, the catalytic light chain of enterokinase, IFNα 2, IL-17²⁰, alkaline phosphatase, AppA¹⁹ and many others. Using this strategy, we expressed different VEGF-C constructs including truncation mutants and the full-length protein (Phe32-Ser419). However, when we analyzed the cytoplasmic fraction of E. coli transformed with these constructs, none showed any biological activity towards VEGFR-3 (Supplementary Fig. S2). Similar to our first attempts, the VEGF-C proteins aggregated into inclusion bodies visible as thick bands in the total cell protein fractions on SDS-PAGE gels (Supplementary Fig. S2).

Periplasmic expression

Because of the unsuccessful results using the CyDisCo system, we turned to periplasmic targeting. Targeting a protein to the periplasmic space, which is a more favorable environment for disulfide bridge formation, has been used to produce active extracellular proteins without the need for additional folding. However, this approach has been most successful with small proteins containing relatively few disulfide bonds, such as IL-3 or IL-4^29,30. We wanted to know whether a similar approach would succeed for a protein like VEGF-C, whose cystine content well exceeds the average for cystine-knot growth factors. We inserted sequences for the pelB signal peptide in front of 4 different cDNAs coding for different forms of mature and full-length VEGF-C. After transformation of these constructs to BL21 (DE3) E. coli, we assayed the periplasmic protein fraction for VEGF-C protein and activity but none of the periplasmic extracts showed any significant biological activity in the Ba/F3-VEGFR-3/EpoR assay (Supplementary Fig. S3).

Using maltose-binding protein as a solubility tag for VEGF-C yields active, mature VEGF-C in E. coli Origami (DE3) cytoplasm

In our previous cytoplasmic expression attempts, VEGF-C aggregated into inclusion bodies independently of whether it was expressed in a redox-modified (strain AD494 with mutation in thioredoxin reductase (ΔtrxB)) or non-redox-modified (in the presence of CyDisCo system) host. Searching for alternative means to prevent the formation of inclusion bodies, we expressed VEGF-C as a fusion protein with maltose-binding protein (MBP), which is known for its ability to enhance the solubility of its fusion partners³¹. We expressed four different forms of MBP-tagged VEGF-C in E. coli strain BL21 (DE3). Although VEGF-C was visible in the soluble cytosolic fraction on SDS-PAGE (Fig. 3a), no activity was observed when testing cytoplasmic extracts for biological activity (Fig. 3b). However, expression of the same MBP-VEGF-C constructs in the cytoplasm of E. coli strain such as Origami (DE3) (mutation in both glutathione reductase (Δgor) and ΔtrxB) facilitated correct disulfide bridge formation and folding of VEGF-C. The cytosolic fraction of the E. coli strain Origami (DE3) expressing the mature forms of MBP-tagged VEGF-C showed activity in the Ba/F3-VEGFR-3/EpoR assay (Fig. 3c, d).

The minimal form of mature VEGF-C showed the strongest response in the Ba/F3-VEGFR-3/EpoR assay and was used for most subsequent assays. We confirmed its biological activity also for the alternative VEGF-C receptor (VEGFR-2) in a bioassay using Ba/F3 cells expressing VEGFR-2/EpoR chimeric receptors (Fig. 4a). This minimal form of mature VEGF-C also successfully stimulated the phosphorylation of VEGFR-2 and VEGFR-3 stably expressed by PAE cells (Fig. 4b).

Characterization of the MBP-tagged VEGF-C

To confirm that the MBP-tag combined with Origami (DE3) strain was responsible for the gain in biological activity, we subcloned the coding sequence for minimal mature VEGF-C from the kanamycin resistance plasmid pET28b(+) to the equivalent ampicillin resistance plasmid pET15b(+) to be able to transform it into the kanamycin-resistant Origami (DE3) cells. To our surprise, also the untagged minimal mature VEGF-C expressed in Origami (DE3) cells showed biological activity in the Ba/F3-VEGFR-3/EpoR assay (Supplementary Fig. S4). However, when we compared the amounts of MBP-tagged and untagged minimal mature VEGF-C from a quantified Western blot, the activity of the untagged protein remained much below the activity of the MBP-tagged protein (Fig. 5).

Further, we purified the MBP-tagged minimal mature VEGF-C by immobilized metal chelate affinity chromatography using Ni sepharose followed by size exclusion chromatography (SEC) (Fig. 6a). We analyzed the purified fractions by SDS-PAGE/Western blotting (Fig. 6b, c) and tested them in the Ba/F3-VEGFR-3/EpoR assay (Fig. 6d). The majority of biological activity was recovered from fraction A10 corresponding to a size of approximately 40 kDa.

Since the majority of the active VEGF-C remained unpurified in the flowthrough of the Ni sepharose affinity chromatography, we further used amylose affinity chromatography followed by cleavage of the MBP tag using TEV protease and then SEC to purify the active VEGF-C (Fig. 7). The bioactivity of the purified fractions was analyzed using Ba/F3-VEGFR-3/EpoR assay, and the majority of bioactivity was contained in fraction A21. Similar level of bioactivity from both MBP-cleaved VEGF-C and uncleaved fusion protein indicated that removal of the MBP tag is not required to recover biological activity of VEGF-C.

No activation of bacterial full-length VEGF-C by the 293T cell secretome

Unsurprisingly, the full-length VEGF-C cDNA, when used for protein production in E. coli, did not yield active protein, because unprocessed, full-length VEGF-C does not activate VEGFR-3⁵, and the specific proteins needed for its activation are not produced by E. coli. Hence, we exposed the bacterial full-length protein to 293T cells, which secrete ADAMTS3 and CCBE1, both of which are sufficient for the activation of pro-VEGF-C⁵. However, this procedure did not increase the biological activity of the full-length VEGF-C as measured by the Ba/F3-VEGFR-3 assay (Supplementary Fig. S5).

Active, mature VEGF-C can also be obtained by folding solubilized inclusion bodies

While getting the first disappointing results in our attempts to directly generate bioactive VEGF-C in the E. coli cyto- or periplasm, we started to evaluate different folding conditions for solubilized inclusion bodies. To obtain biologically active protein, we solubilized the inclusion bodies of the minimal mature form of VEGF-C produced from a truncated, synthetic cDNA using 6M guanidinium chloride (GuHCl) and performed four successive limited screens to identify and optimize suitable folding conditions (see Table S3 for the exact folding conditions). The most efficient folding was obtained under slightly alkaline conditions with high chaotrope and salt concentrations, using reduced/oxidized glutathione as a redox pair (buffer composition: 100 mM Tris pH 8.5, 2 mM EDTA, 550 mM GuHCl, 264 mM NaCl, 11 mM KCl, 10 mM GSH/0.1 mM GSSG).

We further determined that these conditions were also suitable to obtain active mature VEGF-C from truncated cDNAs coding for the naturally occurring major and minor mature forms of VEGF-C³². However, there were clear differences in the ability to fold between these three forms. The minor mature form and the minimal receptor binding domain both appeared to fold much easier compared to the major, mature form. A quantitative comparison of the protein activity (Fig. 8a) with a VEGF-C protein standard produced in insect cells²⁶ showed that the yield was approximately 0.01 mg per liter of E. coli culture, and that only about 0.8‰ of the total bacterial VEGF-C protein could be recovered as biologically active VEGF-C using this refolding and purification scheme (Supplementary Fig. S6). Because of the high purity of the inclusion body preparation (estimated > 70%, see Fig. 2a), and because the majority of misfolded protein precipitated during the dialysis step after the refolding, the active VEGF-C protein could be easily purified in a single step using size exclusion chromatography (Fig. 8b).

Discussion

Recombinant proteins produced in E. coli lack important post-translational modifications of mammalian proteins (most notably glycosylation of secreted proteins). However, using E. coli as an expression host is simpler and often more productive compared to mammalian cells. For its low costs, production in E. coli is the method of choice if the downstream applications do not require eukaryotic-specific posttranslational modifications. Also in the manufacturing of biologicals, E. coli is sometimes used (e.g., for insulin³³). However, not all mammalian proteins do fold correctly in E. coli. Cystine knot proteins, which are virtually unknown in the bacterial kingdom, but have developed in fungi, plants, and animals, are often problematic, and the production of extracellular cystine knot proteins in the E. coli cytoplasm leads mostly to protein aggregation and the formation of inclusion bodies²¹.

The vascular endothelial growth factor (VEGF) family of growth factors is interesting because its members are targets for inhibition, e.g. of tumor-induced blood vessel growth^34,35,36,37, and promotion, e.g. of lymphatic vessel growth in lymphedema³⁸. Inhibition has been mostly achieved by antibodies, which are still almost exclusively produced in mammalian cells for both research and therapeutic purposes, and successful attempts to produce full antibodies in E. coli are sparse³⁹. However, most members of the VEGF growth factors have been produced successfully in E. coli^{10,11,40,41,42,43} but the exact refolding conditions have not been reported in some of these works^12,15. Notably, the successful production of the lymphangiogenic VEGFs (VEGF-C and VEGF-D) has not been reported in E. coli to date. The protein production for structural and functional studies on VEGF-C and VEGF-D has been performed for the last 20 years in insect or mammalian cells^{6,44,45,46,47}. Some commercial suppliers offer VEGF-C produced in E. coli from a truncated cDNA, but the reported biological activities are significantly lower compared to corresponding proteins produced by mammalian hosts⁴⁸. When VEGF-C expression is performed from a truncated cDNA, an odd 9th cysteine in the VEGF homology domain interferes with correct disulfide bond formation^17,49. This cysteine is present in both VEGF-C and VEGF-D but absent from all other VEGFs.

Counter-intuitively, commercially available growth media for lymphatic endothelial cells (LECs) are mostly not supplemented with the lymphangiogenic VEGF-C, but with VEGF-A, which is the primary growth factor for blood vascular endothelial cells (BECs). Unlike VEGF-C, VEGF-A cannot activate the primary lymphangiogenic growth factor receptor VEGFR-3^1,50. Hence, a readily available source of biologically active VEGF-C for lymphatic endothelial cell culture is of interest.

We noticed that in mammalian cells, producing VEGF-C from a full-length cDNA is slower compared to a truncated cDNA, most likely due to the vastly increased possibilities for disulfide bond formation. But irrespective of whether we used a full-length or a truncated cDNA, our initial attempts to produce VEGF-C in bacteria led to inclusion body formation, a typical result for many other cystine knot growth factors^51,52,53. In eukaryotic cells, protein disulfide isomerases (PDIs) are deployed to ensure rapid isomerization into the correct bonding pattern. While many secreted proteins with disulfide bonds have been successfully co-expressed in E. coli with the CyDisCo expression system, we failed to generate bioactive VEGF-C with this strategy.

However, even in the Kringle-2 serine protease fragment vtPA^20,54, which is used as the acid test for disulfide bond formation in E. coli, only 5.1% of the amino acid residues are cysteines, while in the various mature VEGF-C forms the cysteine content is between 7.2 and 8.6%. From the 18 cysteine residues in the dimer, two remain unpaired^16,49,55, and correct inter- and intramolecular disulfide bond formation in VEGF-C is likely a rare event compared to aggregation, even with PDI assistance. A large number of different PDI family members (21, and an even larger number of thioredoxin domain-containing proteins) suggests that specialization has occurred among these proteins. Possibly, with S. cerevisiae ERV1 and H. sapiens PDI of the CyDisCo system, we simply tested the wrong enzymes. The same might have been the case in a previous attempt to improve the quality of VEGF-C by co-expressing CALR, CDC37, PH4B, PDIA3, or PPIB in insect cells⁵⁶. It is also possible that VEGF-C folding benefits from the assistance by a specialized chaperone, as was reported for the closely related VEGF-A⁵⁷. However, even though the redox environment of the bacterial cytoplasm does not per se prevent the formation of disulfide bonds, the typical environment of most PDIs is the eukaryotic endoplasmic reticulum, which features a different redox environment compared to the cytoplasm⁵⁸.

We chose two parallel strategies to produce bioactive VEGF-C: Folding from solubilized inclusion bodies and preventing the formation of inclusion bodies by fusing VEGF-C to the solubility-enhancing maltose-binding protein³¹. The first approach led to the identification of suitable folding conditions, which were similar to conditions previously reported to work for other VEGF family members.

Fusing VEGF-C to the solubility-enhancing MBP was unsuccessful on its own in the E. coli strain BL21, but when combined with the modified cytoplasmic redox-environment of the Origami E. coli strain led to the generation of bioactive VEGF-C. Compared to the soluble VEGF-C in the Origami strain, the insoluble, aggregated VEGF-C in the BL21 strain was largely protected from proteolytic degradation. Proteolytic degradation is at least partly responsible for the low yield of VEGF-C after purification (compare Fig. 3a with Fig. 3c, Figs. 6 and 7). The Western blotting with pentahis-antibody detection indicates that proteolytic processing removes the hexahistidine tag from the MBP-VEGF-C fusion protein. However, VEGF-C binds nevertheless to the Ni sepharose similar to what has been shown previously for VEGF-A⁵⁹. Yet, this binding is not efficient, and majority of active VEGF-C remains in the flowthrough during this step (Fig. 6b–d). VEGF-C recovery could be increased to 0.6 mg/L of bacterial culture using amylose resin, which binds to the MBP tag. However, due to proteolytic processing (see Fig. 7c, e) and/or improper folding of the MBP tag in the Origami strain, a large amount of the active VEGF-C was still present in the flowthrough.

We were surprised to see that bioactive VEGF-C could also be produced in the E. coli Origami strain without the need for the MBP moiety. However, a side-by-side comparison of quantified proteins showed that without the MBP moiety, the activities that we could recover were minimal compared to the MBP-tagged VEGF-C. Such activities were even sometimes undetectable in our experiments depending on the sensitivity of the bioassay. Interestingly, after Ni sepharose affinity chromatography, the major biologic activity was recovered from the fraction containing VEGF-C fused with a partially proteolytically processed MBP. In addition, MBP-tagged VEGF-C eluted from the amylose resin had a very similar level of bioactivity in Ba/F3-VEGFR-3 assay compared to VEGF-C, from which the MBP moeity had been removed by TEV cleavage. This indicates that removal of the MBP tag is not required to recover biological activity of VEGF-C. The X-ray structure of VEGF-C complexed with VEGFR-2 (or VEGFR-3)^16,60 supports the notion that in a VEGF-C/VEGFR-3 complex, an MBP domain N-terminally to the VEGF homology domain would point away from the receptor and thus not interfere with receptor binding and activation. On the other hand, domains C-terminally to the VHD might point towards the cell surface and thus be more likely to interfere with receptor binding and/or activation, similar to the silk-homology domain, which keeps pro-VEGF-C inactive^5,32,61. In the most straightforward scenario, these domains would be located between the extracellular domains 4 and 5 of VEGFR-3, which are instrumental in propagating the dimerization that is initiated by domains 1–3 towards the cell surface and interior⁶⁰.

The total yield of VEGF-C remained modest (about 0.1 and 0.6 mg of active VEGF-C per liter of E. coli culture from Ni sepharose and amylose affinity chromatography, respectively). However, the yield was limited due to incomplete binding of VEGF-C to both resins due to partial or complete cleavage of the N-terminal hexahistidine or the MBP tag, which resulted in the flowthrough showing almost the same level of activity in the Ba/F3-VEGFR-3 assay as the input lysate (Figs. 6d, 7e). We could increase the yield by sixfold using amylose affinity chromatography. We estimate that alternative chromatographic methods such as using immobilized VEGFR-3⁵⁰ could further increase the yield. Together with optimization of the production parameters, the use of E. coli could therefore be a viable replacement for the eukaryotic expression of VEGF-C. Even larger improvements could perhaps be achieved by deploying PDIs in the Origami strain using the MBP-VEGF-C fusion protein, a combination that was not tested in our study.

In addition to producing mature, active VEGF-C from a truncated cDNA, we also attempted to produce full-length VEGF-C (i.e., VEGF-C together with its N- and C-terminal propeptides). However, none of our attempts were successful. We speculate that the repetitive cysteine-rich Balbian Ring 3 protein (BR3) motif in the C-terminal domain of VEGF-C precludes the generation of any meaningful amounts of correctly folded protein. Even if correctly folded, such full-length protein would not be active unless concertedly cleaved by a preprotein convertase such as furin and a specific activating enzyme (ADAMTS3, KLK3, cathepsin D, plasmin, thrombin). Because both cleavages can happen in the supernatant of 293T cells², we exposed the bacterial full-length VEGF-C to conditioned 293T cell supernatant, but could not detect any activation.

VEGF-C is the central molecule required for lymphatic growth. It has potential applications in vascular biology research as a supplement in lymphatic endothelial cell culture, and as a pharmacological agent in the treatment of lymphedema. VEGF-C has already been used—in the form of the adenoviral vector AdVEGF-C—in clinical studies to treat lymphedema³⁸. While bacterial VEGF-C could be a cost-effective recombinant source of the growth factor for many in-vitro applications, glycosylated VEGF-C remains the first choice for in-vivo applications. Although mammalian-type N-glycosylation has been engineered into E. coli⁶², the routine delivery of glycosylated therapeutic proteins is currently achieved using viral vectors, mRNA drugs, or recombinant protein derived from a mammalian cell line.

Materials and methods

Clonings

CyDisCo

To produce the CyDisCo constructs, a synthetic, E. coli-codon-optimized cDNA coding for human VEGF-C (Phe32-Ser419 with mutation Cys137Ala) was used as a PCR template to generate the VEGF-C fragments listed in Table S1. The primers used for the amplification are listed in Table S2. VEGF-C genes were cloned into vector pJV84²⁸ via digestion of parental vector and PCR product with NdeI/BamHI and ligation. All constructs have a C-terminal hexahistidine-tag after the BamHI restriction site, which translates into Gly-Ser.

Periplasmic expression

For periplasmic expression, we inserted the coding sequences for the pelB secretion signal (annealed primers 5′-GATCCGAATTAATTCCGATATCCATGGCCATCGCCGGCTGGGCAGCGAGGAGCAGCAGACCAGCAGCAGCGGTCGGCAGCAGGTATTTCA-3′ and 5′-TATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGATATCGGAATTAATTCG-3′) into BamHI/NdeI-opened pET24b(+) (Novagen), resulting in pET24b(+)-pelB. All VEGF-C cDNAs were transferred from pET28b(+) to pET24b(+)-pelB as StyI fragments, except for the minor, mature form, which was amplified by PCR with primers 5′-CCGCGAAGACTTCATGACAGAAGAGACTATAAAATTTGC-3′ and 5′-CCGGATCCTCAACGTCTAATAATGGAATGAACTTG-3′ and transferred as NcoI/BamHI fragment (Table S1).

MBP-tagged expression

For MBP-tagged VEGF-C expression in the E. coli cytosol, all VEGF-C cDNAs were transferred from the periplasmic expression vector pET24(+)-pelB into a modified pMJ915 vector⁶³. To create this modified expression vector (called pMJ915-MBP), the Cas9 CDS and the bom element of the pMJ915 vector had been deleted. All transfers happened via restriction enzyme cloning of PCR-amplified fragments (for cloning details see Table S1 and the supplemental plasmid maps).

Cytoplasmic expression

To produce the different forms of VEGF-C inclusion bodies in the E. coli cytosol for solubilization and folding, its cDNAs were cloned into pET28b(+) (Novagen; for cloning details see Table S1 and the supplemental plasmid maps).

All DNA constructs generated in this study were verified by Sanger sequencing.

Cell lines

293T cells were obtained from ATCC (RRID: CVCL_0063). Stably transfected PAE cells expressing VEGFR-3⁶⁴ and VEGFR-2⁶⁵ and Ba/F3 cells stably expressing chimeric VEGFR-3⁶⁶ and VEGFR-2⁶⁷ were obtained directly from the reference laboratories. All the cells were grown in D-MEM containing 10% FCS.

Antibodies

We used the following antibodies: anti-VEGF-C antiserum 6 (AS 6)⁶⁸ and antiserum 882 (AS 882)², Penta-His antibody (RRID:AB_2619735), anti-phosphotyrosine antibody PY20, anti-VEGFR-3 antibody 707⁶⁹, anti-VEGFR-2 antibody RS-2⁶⁵.

Pulse chase

293T cells were transfected with the indicated constructs using JetPEI and grown for 36 h on 6-cm dishes to almost-confluency. Cells were starved for 30 min in met-/cys-deficient D-MEM 5% dialyzed FCS, after which 1 mL of labeling medium (met-/cys-deficient D-MEM supplemented with 20 µL EasyTag EXPRESS ³⁵S/mL (Perkin Elmer) and 5% dialyzed FCS) was added to each dish for 2 h. Thereafter, cells were washed with warm PBS and 5 mL of chase medium was added (D-MEM 10% FCS + 1 mM cold L-methionine + 2 mM cold L-cysteine). At the indicated time points dishes were placed on ice, the medium was removed and the cells were washed with PBS and lysed. Analysis was performed by immunoprecipitating VEGF-C with anti-hVEGF-C antiserum⁶⁸. The immunoprecipitated samples were resolved on a reducing SDS-PAGE and signals captured from dried gels with a phosphoimager plate and a Typhoon scanner.

Glycosylation mutants

To produce the single and double glycosylation mutants of VEGF-C, N > Q substitution was done in the cDNA of VEGF-C at amino acid position N175 and/or N205. Equivalent N > W glycosylation mutants were also cloned. 293T cells were transfected with the mutant constructs in 6-well-plates using the calcium phosphate method followed by metabolic labeling. EasyTag EXPRESS ³⁵S was used 8 µL/mL and cells were allowed to incorporate the label for 24 h. The supernatants and lysates were subjected to immunoprecipitation with VEGFR-2/Fc, VEGFR-3/Fc and AS882, respectively. The immunoprecipitated samples were resolved by 15% SDS-PAGE and the image of the dried gels was acquired with a Bas-1500 Bio Imaging Analyzer (Fujifilm).

Protein production

CyDisCo

For CyDisCo expression, chemically competent MG1655 cells were transformed with pAGJ plasmid (AmpR) encoding respective VEGF-C fragments (Table S1) and with a second plasmid pMJS205 carrying CyDisCo components Erv1p and PDI. A single bacterial colony was picked from the transformation agar plate or from a restreaked frozen glycerol stock to inoculate 2 mL of LB containing 0.4% glucose and selection markers. Pre-culture was incubated for about 6 h at 37 °C at 250 rpm. 25 mL of freshly made chemically defined media was prepared in 250 mL shake flasks and inoculated with 250 µL of pre-culture. (One liter of chemically defined media was composed of 3.2 g of NH₄Cl, 3.25 g glucose monohydrate, 11.1 g glycerol, 8.15 g lactose monohydrate, 1.2 g MgSO₄.7H₂O, 17.42 g of K₂HPO₄, 1.86 g of citric acid monohydrate, 100 mg of Fe III citrate, 0.23 mg of Na₂MoO₄·2H₂O, 2.5 mg of CoCl₂·6H₂O, 15 mg of MnCl₂·4H₂O, 1.5 mg of CuCl₂·2H₂O, 3 mg of boric acid, 33.8 mg of zinc acetate.2H₂O, and 12.5 mg of Na₂-EDTA). The shaker flasks were sealed with air-permeable membrane AirOtop Enhanced Seals (Thomson) and the cultures were incubated o/n at 30 °C, 200 rpm (Infors HT incubator). The next day cultures were induced with 0.5 mM IPTG and incubated at 30 °C, 200 rpm for 24 h.

The bacteria were harvested by centrifugation at 5000g for 15 min at 4 °C. The supernatants were discarded, and the bacterial pellets were washed by resuspending in a total of 1/5 to 1/2 of the bacterial culture volume of cold bacterial harvest buffer (50 mM Tris/HCl, 2 mM EDTA, pH 8) followed by centrifugation at 10,000g for 10 min or 20,000 g for 1 min. The cells were then resuspended in 25 mL of lysis buffer (50 mM phosphate buffer, pH = 7.4 with 0.1 mg/mL lysozyme (lysozyme from chicken egg white, Sigma Aldrich) and 20 µg/mL DNase (DNase I from bovine pancreas, Roche)). The lysates were incubated at room temperature for 15 min and frozen at − 20 °C. The lysates were thawed after at least 16 h and total and soluble fractions + /− CyDisCo were resolved on a 12.5% SDS-PAGE gel.

Expression for periplasmic targeting

For periplasmic expression, a 10-mL pre-culture was grown o/n at 37 °C in kanamycin- and tetracycline-supplemented LB medium (50 µg/mL and 12.5 µg/mL, respectively) of a single colony from the transformation agar plate. Subsequently, the o/n culture was diluted 1:100 into 25 mL LB medium in 250-mL Erlenmeyer flasks supplemented with MgSO₄ to a final concentration of 0.5 mM. Bacteria were grown at 37 °C in an orbital shaker (250 rpm) until an OD₆₀₀ of 0.5, and IPTG was added from a 100 mM stock to a final concentration of 100 µM. Incubation was continued for 3 h. Bacteria were harvested as described above for the CyDisCo expression.

The periplasmic extraction was performed based on cold osmotic shock with MgCl₂⁷⁰. Briefly, the bacterial pellet was washed with 85 µL PBS, centrifuged for 3 min at 20,000g and the supernatant was carefully removed. The cell pellet was then resuspended in 900 µL of cold spheroplast buffer (0.1 M Tris/HCl, 500 mM sucrose, 0.5 mM EDTA, pH 8). After 5 min incubation on ice and 3 min centrifugation at 20,000g at 4 °C, the supernatant was discarded. The bacterial pellet was then resuspended in 400 µL of cold 1 mM MgCl₂ and after 15 s incubation on ice, 20 µL MgSO₄ (20 mM) was added. Finally, centrifugation was performed as above and the periplasmic fraction (supernatant) was collected and stored at − 20 °C.

Cytoplasmic expression

E. coli Origami (DE3) and BL21 (DE3) cells were transformed with the pET15b + or pMJ915 and pET28b + or pMJ915 expression plasmids, respectively. Single bacterial colonies were picked from the transformation agar plate and 5 mL o/n cultures were grown in LB medium with the corresponding antibiotic(s): 100 µg/mL ampicillin + 50 µg/mL kanamycin + 12.5 µg/mL tetracycline for Origami (DE3), and 50 µg/mL kanamycin for BL21 (DE3). The o/n cultures were diluted 1:100 into Terrific Broth (TB) medium. For analytical expression, 10-mL-cultures were grown in 50-mL Falcon tubes. Bacteria were grown at 37 °C in an orbital shaker (250 rpm) until an OD₆₀₀ of 0.4–0.8, and IPTG was added from a 100 mM stock to a final concentration of 0.5 mM. Incubation was continued for 20 h at 25 °C. Bacteria were harvested as described above for the CyDisCo expression.

Following protein production, the frozen E. coli were thawed on ice and resuspended in 1 mL PBS. After mixing 100 µL of the cell suspension with 100 µL of 2×Laemmli sample buffer, cells were sonicated in an ice-water bath (Fisher Scientific FB120, 1/8 in. tip, 8 cycles of 6 s/10 s burst/cooling at 50% amplitude). The bacterial lysate was centrifuged at 13,000g for 30 min at 4 °C and the supernatant and cell pellets were collected and stored at − 20 °C.

Cytoplasmic expression for refolding

A single bacterial colony was picked from the transformation agar plate or a restreaked frozen glycerol stock and a 5 mL o/n culture was grown in LB medium containing 50 µg/mL kanamycin. The o/n culture was diluted 1:100–1:1000 into Terrific Broth (TB) medium. For analytical batches, 10-mL-cultures were grown in 50-mL Falcon tubes, and for production batches, 250-mL-cultures were grown in 1 L Erlenmeyer flasks. Bacteria were grown at 37 °C in an orbital shaker (250 rpm) until an OD₆₀₀ of 0.4–0.8, and IPTG was added from a 100 mM stock to a final concentration of 1 mM. Incubation was continued for 3.5 to 4 h. Bacteria were harvested as described above for the CyDisCo expression and stored at − 80 °C.

Bacterial lysis for refolding

Following protein production, the thawed E. coli pellet was washed by resuspending it in bacterial harvest buffer (25 mL for each gram of bacterial pellet, but maximally 50 mL) and centrifuging at 8000g for 15 min at 4 °C. After this, they were lysed in bacterial lysis buffer (50 mM Tris/HCl pH 8.0, 2 mM EDTA, 10 mM ß-mercaptoethanol, 5% glycerol; 5 mL for each gram of bacterial pellet), which was complemented with lysozyme (1 mg lysozyme to each gram of bacterial pellet from a 10 mg/mL lysozyme solution in bacterial harvest buffer), and the mixture was stirred at RT for 20 min.

After this, 1/9 volume of the detergent mix (10% NP-40 and 0.5% NaDOC) was added to the mixture (565 µL for each gram bacterial pellet), and the stirring was continued for another 20 min. Then 250 units of DNaseI were added for each gram bacterial pellet and stirring continued for 30 min at 30 °C. Cells were then sonicated in an ice-water bath (Fisher Scientific FB120, 1/8 in. tip, 3–6 cycles of 10 s/20 s burst/cooling at 100% amplitude). The maximal volume for sonication was 8 mL; larger volumes were split into aliquots and sonicated separately.

Inclusion body preparation

The bacterial lysate was centrifuged at 13,000g for 30 min at 4 °C. The pellet was resuspended in bacterial lysis buffer. The lysozyme, DNaseI, and detergent treatments and centrifugation were repeated up to 4 times. To remove lysozyme, DNaseI, and detergent, the inclusion body pellet was three times resuspended in bacterial lysis buffer and centrifuged at 13,000g (except for the last centrifugation, which was performed at 10,000g). For storage, inclusion body pellets were frozen at − 70 °C before the last centrifugation step.

Solubilization of inclusion bodies

Each gram of isolated inclusion bodies was resuspended in 4 mL of solubilization buffer (6M Gu-HCl, 20 mM Tris/HCl pH 8.0, 5 mM EDTA, 10 mM DTT) and incubated for 2 h at RT under agitation. To remove unsolubilized material, the reaction was centrifuged using an SW41Tl rotor at 20,000 rpm (~ 46,000g) for 30 min and the supernatant was transferred to a new tube.

Protein folding

In the first screen, we tested 32 different conditions for their ability to support the folding of a minimal form of VEGF-C, which consisted only of the essential VHD. Half of these conditions were identical to the ones reported by⁷¹, the rest were identical to reportedly working folding conditions for other proteins from the same (PDGF/VEGF) protein family (see Table S3a). Based on the two most efficient folding conditions, we set up two additional screens to optimize the conditions (Tables S3b and S3c). A fourth folding screen was then performed using selected conditions from the previous screens (see Table S3d), but this time we used both the minimal version of VEGF-C as well as the two most abundant naturally occurring forms of mature VEGF-C: the major, mature form (produced by furin- and ADAMTS3-cleavage) and the minor, mature form (produced by furin- and plasmin-cleavage) (for a review about the different VEGF-C cleavages, see⁷²). Refolding was performed by adding 50 µL of solubilized inclusion bodies to 1 mL refolding buffer and incubating the reaction at + 4 °C under gentle agitation o/n. After refolding, samples were dialyzed against three buffer changes of 100 volumes of PBS-T (0.05% Tween 20) and a final buffer change against PBS. Protein that had precipitated during dialysis was removed by centrifugation at 20,800g for 30 min.

Assays for biologically active, correctly folded VEGF-C

For the first three folding screens, we assayed the successful refolding by testing the ability of the samples to induce phosphorylation of the main VEGF-C receptor (VEGFR-3) as described previously⁴⁶. In a nutshell, serum-starved PAE cells stably transfected with VEGFR-3 were exposed to a 1:10 dilution of the refolded samples for 5 min, after which the cells were placed on ice, lysed, and analyzed for phosphorylated VEGFR-3 after polyacrylamide gel electrophoresis and Western blotting (data not shown). The refolded samples were used at a concentration of 10%.

The samples of the fourth folding screen were assayed using a chimeric receptor consisting of the extracellular domain of human VEGFR-3 fused to the transmembrane and intracellular domain of the erythropoietin receptor (hVEGFR-3/EpoR) as described previously⁶⁶. Since we had never found any discrepancies between the phosphorylation assays using native VEGFR-3 and the Ba/F3-hVEGFR-3/EpoR bioassays when comparing dimeric VEGF growth factors^6,46, we used the chimeric bioassay for the rest of these studies, notably for assaying the VEGF-C targeted to the periplasm and the MBP-tagged VEGF-C.

Purification of the refolded VEGF-C

The dialyzed and cleared refolded material was concentrated on a YM-3 Centricon device according to the instructions of the manufacturer (Millipore) and resolved on a Superdex 75 10/300 GL size exclusion chromatography column using PBS as running buffer and a flow rate between 0.5 and 0.6 mL/min. Calibration of the column was performed with a blend of BSA (New England Biolabs, #B7000), carbonic anhydrase (Sigma C7025) and RNaseA (Qiagen #19101) in PBS (each protein at 1.25 mg/mL). The dimeric (11.5–13.5 mL) as well as the monomeric (13.5–15.5 mL) fractions showed both biological activity in the Ba/F3-VEGFR-3/EpoR assay.

Purification of the MBP/VEGF-C fusion protein

MBP-tagged minimal mature VEGF-C in the modified pMJ915 vector was transformed into E. coli Origami (DE3) cells and a single bacterial colony was picked from the transformation agar plate and a 10 mL o/n culture was grown in LB medium containing 100 µg/mL ampicillin, 50 µg/mL kanamycin, and 12.5 µg/mL tetracycline. The o/n culture was diluted 1:100 into 1 L of LB medium. Bacteria were grown at 37 °C in an orbital shaker (250 rpm) until an OD₆₀₀ of 0.4–0.8 and induced with 0.5 mM IPTG. Incubation was continued for 20 h at 25 °C. Bacteria were harvested as described above for the CyDisCo expression.

Following protein production, the thawed bacterial pellet was resuspended in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl) complemented with 0.1 mg/mL lysozyme and protease inhibitor (Pierce™ Protease Inhibitor Tablets, EDTA-free), and kept on ice for 30 min. Cells were sonicated in an ice-water bath (Fisher Scientific FB120, ¼ inch tip, 8–10 cycles of 10 s/20 s burst/cooling at 100% amplitude). The bacterial lysate was centrifuged at 13,000g for 30 min at 4 °C and the supernatant was collected for purification.

For the purification of his-tagged proteins, 1 mL of Ni sepharose (Excel, Cytiva) was added to the lysate for o/n batch binding at 4 °C. The Ni sepharose was loaded to a column, washed with 10 mM imidazole, and eluted with 500 mM imidazole. For amylose affinity purification, 1 mL of amylose resin (NEB) was added to the lysate for o/n batch binding. The amylose resin was then washed 5 times with binding buffer and eluted with 10 mM maltose. The peak fraction or concentrated peak fractions (500 µL) were further resolved on a Superdex 200 10/300 GL size exclusion chromatography column using PBS as a running buffer and a flow rate between 0.4 and 0.5 mL/min. The concentrated fractions after elution with maltose were treated with 5 µL TEV protease (2.4 mg/mL; purified in-house) to remove the MBP tag before SEC. The fractions were analyzed by SDS-PAGE followed by Coomassie/silver staining and Western blotting. The biological activity of the resolved fractions was analyzed using Ba/F3-VEGFR-3/EpoR assay.

Activation of bacterial full-length VEGF-C using 293T cells

293T cells on a 12-well-plate were 70% confluent when the medium was changed to 400 µL E. coli cell lysates that were filtered through 0.45 µm syringe filter units. The lysates were obtained by sonication of Origami (DE3) resuspended in serum-free D-MEM. 24 h later the conditioned media were collected and supernatants were used in Ba/F3 assay at a maximal concentration of 20% of final culture volume.

Statistical analysis

Data are presented as mean ± SD. Data were analyzed using GraphPad Prism statistical analysis software (version 8.4.2 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com), OriginPro for statistical analysis (version 9.7), and Numbers (version 10.3.9). The number of replicates is mentioned in the respective figure legends.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary figures and tables.

References

Rauniyar, K., Jha, S. K. & Jeltsch, M. Biology of vascular endothelial growth factor C in the morphogenesis of lymphatic vessels. Front. Bioeng. Biotechnol. 6, 7 (2018).
Article PubMed PubMed Central Google Scholar
Joukov, V. et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. 16, 3898–3911 (1997).
Article CAS PubMed PubMed Central Google Scholar
Siegfried, G. et al. The secretory proprotein convertases furin, PC5, and PC7 activate VEGF-C to induce tumorigenesis. J. Clin. Invest. 111, 1723–1732 (2003).
Article CAS PubMed PubMed Central Google Scholar
McColl, B. K. et al. Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D. J. Exp. Med. 198, 863–868 (2003).
Article CAS PubMed PubMed Central Google Scholar
Jeltsch, M. et al. CCBE1 enhances lymphangiogenesis via a disintegrin and metalloprotease with thrombospondin motifs-3–mediated vascular endothelial growth factor-C activation. Circulation 129, 1962–1971 (2014).
Article CAS PubMed Google Scholar
Jha, S. K. et al. KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D. eLife 8, e44478 (2019).
Article PubMed PubMed Central Google Scholar
Lim, L. et al. Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC. Blood 134, 1764–1775 (2019).
Article PubMed PubMed Central Google Scholar
Kase, S. et al. αB-crystallin regulation of angiogenesis by modulation of VEGF. Blood 115, 3398–3406 (2010).
Article CAS PubMed PubMed Central Google Scholar
Ozawa, K. et al. Expression of the oxygen-regulated protein ORP150 accelerates wound healing by modulating intracellular VEGF transport. J. Clin. Invest. 108, 41–50 (2001).
Article CAS PubMed PubMed Central Google Scholar
Christinger, H. W. et al. Crystallization of the receptor binding domain of vascular endothelial growth factor. Proteins Struct. Funct. Bioinform. 26, 353–357 (1996).
Article CAS Google Scholar
Christinger, H. W., Fuh, G., de Vos, A. M. & Wiesmann, C. The crystal structure of placental growth factor in complex with domain 2 of vascular endothelial growth factor receptor-1. J. Biol. Chem. 279, 10382–10388 (2004).
Article CAS PubMed Google Scholar
Iyer, S. et al. The crystal structure of human placenta growth factor-1 (PlGF-1), an angiogenic protein, at 2.0 Å resolution. J. Biol. Chem. 276, 12153–12161 (2001).
Article CAS PubMed Google Scholar
Iyer, S., Scotney, P. D., Nash, A. D. & Ravi Acharya, K. Crystal structure of human vascular endothelial growth factor-B: Identification of amino acids important for receptor binding. J. Mol. Biol. 359, 76–85 (2006).
Article CAS PubMed Google Scholar
Muller, Y. A. et al. Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site. Proc. Natl. Acad. Sci. USA 94, 7192–7197 (1997).
Article ADS CAS PubMed PubMed Central Google Scholar
Scrofani, S. D., Fabri, L. J., Xu, P., Maccarone, P. & Nash, A. D. Purification and refolding of vascular endothelial growth factor-B. Protein Sci. Publ. Protein Soc. 9, 2018–2025 (2000).
Article CAS Google Scholar
Leppänen, V.-M. et al. Structural determinants of growth factor binding and specificity by VEGF receptor 2. Proc. Natl. Acad. Sci. 107, 2425–2430 (2010).
Article ADS PubMed PubMed Central Google Scholar
Leppänen, V.-M. et al. Structural determinants of vascular endothelial growth factor-D receptor binding and specificity. Blood 117, 1507–1515 (2011).
Article PubMed Google Scholar
Bessette, P. H., Åslund, F., Beckwith, J. & Georgiou, G. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. 96, 13703–13708 (1999).
Article ADS CAS PubMed PubMed Central Google Scholar
Hatahet, F., Nguyen, V. D., Salo, K. E. & Ruddock, L. W. Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microb. Cell Factories 9, 67 (2010).
Article Google Scholar
Nguyen, V. D. et al. Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E. coli. Microb. Cell Factories 10, 1 (2011).
Article CAS Google Scholar
de Marco, A. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb. Cell Factories 8, 26 (2009).
Article Google Scholar
Berkmen, M. Production of disulfide-bonded proteins in Escherichia coli. Protein Expr. Purif. 82, 240–251 (2012).
Article CAS PubMed Google Scholar
Ruano-Gallego, D., Fraile, S., Gutierrez, C. & Fernández, L. Á. Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb. Cell Factories 18, 47 (2019).
Article Google Scholar
Matos, C. F. R. O. et al. Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains. Biotechnol. Prog. 30, 281–290 (2014).
Article CAS PubMed Google Scholar
Rasmussen, J. R. Effect of glycosylation on protein function. Curr. Opin. Struct. Biol. 2, 682–686 (1992).
Article CAS Google Scholar
Karpanen, T. et al. Functional interaction of VEGF-C and VEGF-D with neuropilin receptors. FASEB J. 20, 1462–1472 (2006).
Article CAS PubMed Google Scholar
Derman, A. I., Prinz, W. A., Belin, D. & Beckwith, J. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262, 1744–1747 (1993).
Article ADS CAS PubMed Google Scholar
Gaciarz, A. et al. Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli. Microb. Cell Factories 15, 22 (2016).
Article Google Scholar
Dagar, V. K., Adivitiya & Khasa, Y. P. High-level expression and efficient refolding of therapeutically important recombinant human Interleukin-3 (hIL-3) in E. coli. Protein Expr. Purif. 131, 51–59 (2017).
Article CAS PubMed Google Scholar
Lundell, D. et al. Cytoplasmic and periplasmic expression of a highly basic protein, human interleukin 4, in Escherichia coli. J. Ind. Microbiol. 5, 215–227 (1990).
Article CAS PubMed Google Scholar
Lebendiker, M. & Danieli, T. Purification of proteins fused to maltose-binding protein. Methods Mol Biol. 1485, 257–273 (2017).
Article CAS PubMed Google Scholar
Joukov, V. et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. 16, 3898–3911 (1997).
Article CAS PubMed PubMed Central Google Scholar
Riggs, A. D. Making, cloning and expression of human insulin genes in bacteria: The path to Humulin. Endocr. Rev. 25, 374–380 (2021).
Article Google Scholar
Jayson, G. C., Kerbel, R., Ellis, L. M. & Harris, A. L. Antiangiogenic therapy in oncology: current status and future directions. Lancet 388, 518–529 (2016).
Article CAS PubMed Google Scholar
Toi, M. et al. Significance of vascular endothelial growth factor (VEGF)/soluble VEGF receptor-1 relationship in breast cancer. Int. J. Cancer 98, 14–18 (2002).
Article CAS PubMed Google Scholar
Demirovic, A. et al. Targeting human cancer cells with VEGF receptor-2-directed liposomes. Oncol. Rep. 13, 319–324 (2005).
CAS Google Scholar
Bainbridge, J. W. B. et al. A peptide encoded by exon 6 of VEGF (EG3306) inhibits VEGF-induced angiogenesis in vitro and ischaemic retinal neovascularisation in vivo. Biochem. Biophys. Res. Commun. 302, 793–799 (2003).
Article CAS PubMed Google Scholar
Hartiala, P. et al. Phase 1 LymfactinⓇ Study: short-term safety of combined adenoviral VEGF-C and lymph node transfer treatment for upper extremity lymphedema. J. Plast. Reconstr. Aesthet. Surg. 73, 1612–1621 (2020).
Article PubMed Google Scholar
Mazor, Y., Van Blarcom, T., Mabry, R., Iverson, B. L. & Georgiou, G. Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Nat. Biotechnol. 25, 563–565 (2007).
Article CAS PubMed Google Scholar
Pizarro, S. A. et al. High-yield expression of human vascular endothelial growth factor VEGF165 in Escherichia coli and purification for therapeutic applications. Protein Expr. Purif. 72, 184–193 (2010).
Article CAS PubMed Google Scholar
Siemeister, G. et al. Expression of biologically active isoforms of the tumor angiogenesis factor VEGF in Escherichia coli. Biochem. Biophys. Res. Commun. 222, 249–255 (1996).
Article CAS PubMed Google Scholar
Kim, Y. S. et al. Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8. Microb. Cell Factories 20, 9 (2021).
Article CAS Google Scholar
Seyedarabi, A., Cheng, L., Zachary, I. & Djordjevic, S. Production of soluble human vascular endothelial growth factor VEGF-A165-heparin binding domain in Escherichia coli. PLoS ONE 8, e55690 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Oh, S.-J. et al. VEGF and VEGF-C: Specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev. Biol. 188, 96–109 (1997).
Article CAS PubMed Google Scholar
Achen, M. G. et al. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc. Natl. Acad. Sci. 95, 548–553 (1998).
Article ADS CAS PubMed PubMed Central Google Scholar
Jeltsch, M. et al. Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns. J. Biol. Chem. 281, 12187–12195 (2006).
Article CAS PubMed Google Scholar
Davydova, N. et al. Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies. Protein Expr. Purif. 82, 232–239 (2012).
Article CAS PubMed Google Scholar
BioVision. Datasheet: Recombinant Human VEGF-C (2010).
Chiu, J., Wong, J. W. H., Gerometta, M. & Hogg, P. J. Mechanism of dimerization of a recombinant mature vascular endothelial growth factor C. Biochemistry 53, 7–9 (2014).
Article CAS PubMed Google Scholar
Joukov, V. et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J. 15, 290–298 (1996).
Article CAS PubMed PubMed Central Google Scholar
von Einem, S., Schwarz, E. & Rudolph, R. A novel TWO-STEP renaturation procedure for efficient production of recombinant BMP-2. Protein Expr. Purif. 73, 65–69 (2010).
Article Google Scholar
Reigstad, L. J. et al. Platelet-derived growth factor (PDGF)-C, a PDGF family member with a vascular endothelial growth factor-like structure. J. Biol. Chem. 278, 17114–17120 (2003).
Article CAS PubMed Google Scholar
Tuan, T.-L. et al. Engineering, expression and renaturation of targeted TGF-beta fusion proteins. Connect. Tissue Res. 34, 1–9 (1996).
Article CAS PubMed Google Scholar
Obukowicz, M. G. et al. Secretion of active kringle-2-serine protease in Escherichia coli. Biochemistry 29, 9737–9745 (1990).
Article CAS PubMed Google Scholar
Leppanen, V.-M. et al. Structural determinants of vascular endothelial growth factor-D receptor binding and specificity. Blood 117, 1507–1515 (2011).
Article CAS PubMed Google Scholar
Uusitalo, E. Improvement of the Quality of Insect-Cell-Derived, Recombinant Pro-VEGF-C (2015).
Kaplan, O. et al. Enhanced mitogenic activity of recombinant human vascular endothelial growth factor VEGF121 expressed in E. coli Origami B (DE3) with molecular chaperones. PLOS ONE 11, e0163697 (2016).
Article PubMed PubMed Central Google Scholar
Go, Y.-M. & Jones, D. P. Redox compartmentalization in eukaryotic cells. Biochim. Biophys. Acta 1780, 1273–1290 (2008).
Article CAS PubMed PubMed Central Google Scholar
Mohanraj, D., Olson, T. & Ramakrishnan, S. A novel method to purify recombinant vascular endothelial growth factor (VEGF121) expressed in yeast. Biochem. Biophys. Res. Commun. 215, 750–756 (1995).
Article CAS PubMed Google Scholar
Leppänen, V.-M. et al. Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation. Proc. Natl. Acad. Sci. 110, 12960–12965 (2013).
Article ADS PubMed PubMed Central Google Scholar
Jha, S. K. et al. Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1. Sci. Rep. 7, 4916 (2017).
Article ADS PubMed PubMed Central Google Scholar
Valderrama-Rincon, J. D. et al. An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat. Chem. Biol. 8, 434–436 (2012).
Article CAS PubMed PubMed Central Google Scholar
Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).
Article PubMed PubMed Central Google Scholar
Pajusola, K. et al. Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors. Oncogene 9, 3545–3555 (1994).
CAS PubMed Google Scholar
Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M. & Heldin, C. H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J. Biol. Chem. 269, 26988–26995 (1994).
Article CAS PubMed Google Scholar
Achen, M. G. et al. Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3. Eur. J. Biochem. 267, 2505–2515 (2000).
Article CAS PubMed Google Scholar
Stacker, S. A. et al. A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability. J. Biol. Chem. 274, 34884–34892 (1999).
Article CAS PubMed Google Scholar
Baluk, P. et al. Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation. J. Clin. Invest. 115, 247–257 (2005).
Article CAS PubMed PubMed Central Google Scholar
Pajusola, K., Aprelikova, O., Armstrong, E., Morris, S. & Alitalo, K. Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxy terminal tails are produced by alternative processing of primary transcripts. Oncogene 8, 2931–2937 (1993).
CAS PubMed Google Scholar
Malherbe, G., Humphreys, D. P. & Davé, E. A robust fractionation method for protein subcellular localization studies in Escherichia coli. Biotechniques 66, 171–178 (2019).
Article CAS PubMed Google Scholar
Chen, G.-Q. & Gouaux, E. Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli: Application of a novel protein folding screen. Proc. Natl. Acad. Sci. USA 94, 13431–13436 (1997).
Article ADS CAS PubMed PubMed Central Google Scholar
Lackner, M., Schmotz, C. & Jeltsch, M. The proteolytic activation of vascular endothelial growth factor-C. Lymphol. Forsch. Prax. 23, 88–98 (2019).
Google Scholar

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Acknowledgements

We thank Andrea Díaz Iglesias for making excellent competent E. coli cells and Kenny Mattonet for purifying TEV protease.

Funding

The research has been funded by grants from the Jane and Aatos Erkko Foundation, the Päivikki and Sakari Sohlberg Foundation, Novo Nordisk Foundation (no. 21036), and the Academy of Finland (nos. 337430 and 337120). K.R. was supported by the Päivikki and Sakari Sohlberg Foundation and M.J. was supported by the Paulo Foundation and the Einar and Karin Stroem Foundation for Medical Research.

Author information

These authors contributed equally: Khushbu Rauniyar and Soheila Akhondzadeh.

Authors and Affiliations

Drug Research Program, Faculty of Pharmacy, Biocenter 2, University of Helsinki, P.O.B. 56 (Viikinkaari 5E), 00014, Helsinki, Finland
Khushbu Rauniyar, Soheila Akhondzadeh, Jaana Künnapuu & Michael Jeltsch
Individualized Drug Therapy Research Program, University of Helsinki, Helsinki, Finland
Anna Gąciarz & Michael Jeltsch
Wihuri Research Institute, Helsinki, Finland
Michael Jeltsch

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Khushbu Rauniyar
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Soheila Akhondzadeh
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Jaana Künnapuu
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Michael Jeltsch
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K.R., S.A. and M.J. designed and conducted experiments, acquired and analyzed data, and wrote the manuscript. A.G. and J.V. conducted experiments, acquired, analyzed data and contributed parts of the manuscript. M.J. provided critical input to the experimental design and supervised the project. All authors reviewed the manuscript.

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Correspondence to Michael Jeltsch.

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Rauniyar, K., Akhondzadeh, S., Gąciarz, A. et al. Bioactive VEGF-C from E. coli. Sci Rep 12, 18157 (2022). https://doi.org/10.1038/s41598-022-22960-0

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Received: 20 June 2022
Accepted: 21 October 2022
Published: 28 October 2022
DOI: https://doi.org/10.1038/s41598-022-22960-0

This article is cited by

Expansion and collapse of VEGF diversity in major clades of the animal kingdom
- Khushbu Rauniyar
- Honey Bokharaie
- Michael Jeltsch
Angiogenesis (2023)

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Subjects

Abstract

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Introduction

Results

The cysteine-rich C-terminal domain of VEGF-C appears to be responsible for the slow secretion of VEGF-C

Partial lack of N-linked glycosylation does not preclude VEGF-C binding to VEGFR-3

VEGF-C expressed in the cytoplasm of E. coli BL21 (DE3) is found exclusively in inclusion bodies

Coexpression with a sulfhydryl oxidase and disulfide bond isomerase (CyDisCo system) does not yield active VEGF-C

Periplasmic expression

Using maltose-binding protein as a solubility tag for VEGF-C yields active, mature VEGF-C in E. coli Origami (DE3) cytoplasm

Characterization of the MBP-tagged VEGF-C

No activation of bacterial full-length VEGF-C by the 293T cell secretome

Active, mature VEGF-C can also be obtained by folding solubilized inclusion bodies

Discussion

Materials and methods

Clonings

CyDisCo

Periplasmic expression

MBP-tagged expression

Cytoplasmic expression

Cell lines

Antibodies

Pulse chase

Glycosylation mutants

Protein production

CyDisCo

Expression for periplasmic targeting

Cytoplasmic expression

Cytoplasmic expression for refolding

Bacterial lysis for refolding

Inclusion body preparation

Solubilization of inclusion bodies

Protein folding

Assays for biologically active, correctly folded VEGF-C

Purification of the refolded VEGF-C

Purification of the MBP/VEGF-C fusion protein

Activation of bacterial full-length VEGF-C using 293T cells

Statistical analysis

Data availability

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

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Additional information

Publisher's note

Supplementary Information

Supplementary Information 1.

Supplementary Information 2.

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Expansion and collapse of VEGF diversity in major clades of the animal kingdom

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