Production of Human MANF protein
QMCF Technology protein expression system comprises of genetically modified eukaryotic cell line and expression plasmids stably replicating in these cells. The cell line used for protein production are modified suspension CHO (CHOEBNALT85) cells which express two additional proteins for providing replication initiation and effective maintenance of the expression vectors. The replication initiation function is provided by mouse polyomavirus Large T antigen. The stable maintenance function for the vector is provided by the Epstein-Barr virus EBNA1 protein, which by acting on the FR-element provides chromatin attachment and segregation/partitioning function to the expression vector in mitosis and cell division.
QMCF expression plasmids consist of two viral-originated DNA elements for replication and maintenance of the plasmids in dividing cells. The replication of the plasmid is assured by Py minimal replication origin. For maintenance of the QMCF plasmids FR of EBV is used which consists of multiple binding sequences for EBV EBNA-1. DNA binding domains of EBV EBNA-1 interact specifically with appropriate binding sequences in plasmids and non-specifically with chromatin DNA using second DNA binding domains. These interactions assure stable maintenance and partitioning of the expression plasmid during mitosis.
Expression plasmid construction
For production of hMANF protein hEF1-HTLV
promoter-containing plasmid was used (Figure 1). hMANF-encoding cDNA was
codon-optimized and synthesized and cloned into the expression vector.
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Figure 1. Schematic representation of hMANF expression vector. Maintenance sequence (Epstein-Barr Virus Family of Repeats (FR)); PyV core origin – murine polyomavirus origin of replication; SV40 pr – SV 40 promoter controlling expression of Neo resistance gene; Neo/Km – Neomycin/Kanamycin resistance marker; PolyA – polyadenylation sequence; hEF1α-HTLV promoter – human elongation factor 1α promoter with Human T-lymphotrophic virus leader sequence; hMANF – human MANF-encoding cDNA
Transfection, antibiotic selection and protein expression
6x106 exponentially growing CHOEBNALT85 cells (viability 97%) were transfected (BioRad Gene Pulser) with 1 µg of hMANF-expression plasmid.Transfection efficiency of the QMCF-CHO cells was approximately 85%. Cell culture growing details and viability is represented below in Figure 3. To select plasmid-containing cell population, G418 was added 48 hours after transfection. 72 hours after transfection MANF expression was tested from the supernatant of the cells by Western blot analysis using anti-hMANF monoclonal antibody (Figure 2).
Figure 2. Western blot analysis of early-stage expression of MANF using CHOEBNALT85 cells. Line 1. Cell lysate of CHOEBNALT85 [hEF1α-HTLV-hMANF]; Line 2. Supernatant (10µl) of CHOEBNALT85 [hEF1α-HTLV-hMANF]; Line 3. Protein ladder (PageRuler Prestained protein ladder, Fermentas).
The cell culture volume was 200 ml, the culture was repeatedly diluted to hold cell culture at similar volume. 10 days after transfection the antibiotic selection was ended and production cell bank was generated (5 vials 1x107 cells/vial). For production of the protein, 11 days after transfection temperature of the culture was decreased to 30°C, and the culture was additionally fed. Feeding of the culture was repeated every third day. 9 days after start of production, the culture was harvested, the supernatant was filtrated and stored at -20°C prior to purification. After production of the protein, the protein expression was tested by Western blot analysis.
Figure 3. Growth of the hMANF-expressing CHOEBNALT85 cell culture. 48h after transfection G418 was added. 72h after transfection pilot production analysis was performed. 10 days after transfection expression cell bank was generated. From day 11 to 19 the production phase was performed. Temperature was reduced to 30°C, additional nutrients were added to the medium. The viability of the cell culture was more than 85% during antibiotic selection and production. At day 19 after transfection, when viability of the culture starts to decline, supernatant of the culture was clarified by centrifugation and filtered through 0.45 µm filter and stored at -20°C prior to purification.
Figure 4. Western blot analysis of MANF expression after production. Line 1. Cell lysate of CHOEBNALT85 [hEF1α-HTLV-hMANF]; Line 2. Supernatant (10µl) of CHOEBNALT85 [hEF1α-HTLV-hMANF]; Line 3. Protein ladder (PageRuler Prestained protein ladder, Fermentas)
Purification and purity analysisTwo-step ion-exchange chromatography and gel filtration was used for purification of MANF. After purification, protein concentration was measured by BCA Protein Assay Kit (Thermo Scientific, Pierce Protein Research Products). BSA (Pierce) was used as a standard. Purity of the protein was analyzed by coomassie-stained SDS-PAGE electrophoresis (Figure 5) and mass-spectormetry (Figure 6).
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Figure 5. SDS-PAGE analysis of purified human MANF visualized by PageBlue protein staining solution (Fermentas). Line 1. Protein size marker (PageRuler Prestained protein ladder, Fermentas); Line 2. 10 µg of purified human MANF.
Figure 6. Mass-spectrometry analysis of purified human MANF. Mass-spectrometry analysis revealed purified hMANF as pure and homogenous material.
Testing of MANF biological activity
The biological activity of the human recombinant MANF has been tested in two models. In rat 6-hydroxydopamine (6-OHDA) Parkinson’s disease model MANF can protect and also repair dopamine nigrostriatal neurons when injected 6 hours before 6-OHDA or 4 weeks after 6-OHDA. The methods of the MANF testing are described by Lindholm et al., 2007 and Voutilainen et al., 2009.
In the second model, recombinant human MANF protein can rescue sympathetic superior cervical ganglion neurons in culture from etoposide- or staurosporine-induced apoptotic death when injected into the cytoplasm of these neurons. The bioassay of MANF on sympathetic neurons is described by Hellmann et al., 2011.
- Lindholm P, Voutilainen MH, Lauren J, Peranen J, Leppänen VM, Andressoo JO, Lindahl M, Janhunen S, Kalkkinen N, Timmusk T, Tuominen RK, Saarma M (2007) Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature 448: 73-77
- Voutilainen MH, Bäck S, Porsti E, Toppinen L, Lindgren L, Lindholm P, Peränen J, Saarma M*, Tuominen RK (2009) Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of parkinson's disease. J Neurosci 29, 9651-9659 (* corresponding author)
- Hellman M, Arumäe U, Yu LY, Lindholm P, Peränen J, Saarma M*, Permi P* (2011) Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons. J Biol Chem 286: 2675-2680. * Equal contribution.