Case study

Production of human MANF protein

Case study

Technology overview

QMCF Technology comprises of genetically modified eukaryotic cell line and expression plasmids that stably replicate and maintain in these cells. 

QMCF cell lines are stable suspension or adherent cells (for instance CHOEBNALT85) into which two genes have been introduced in order to provide replication initiation and effective maintenance functions to appropriate QMCF expression vectors. The replication initiation function is provided by mouse polyomavirus Large T antigen, and the stable maintenance function for the vector is provided by the Epstein-Barr virus EBNA-1 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. There is a large set of QMCF plasmids that in addition to the regulatory maintenance and replication elements contain different expression cassettes and allow to design protein production experiment in most proper manner. 


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. 


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 – SV40 promoter controlling expression of Neo resistance gene; 
Neo/Km – Neomycin/Kanamycin resistance marker; PolyA – polyadenylation sequence; 
hEF1α-HTLV promoter; 
hMANF – human MANF encoding cDNA


Transfection, antibiotic selection and expression

6x10^6 exponentially growing CHOEBNALT85 cells (viability 97%) were transfected (Bio-Rad Gene Pulser) with 1 µg of hMANF expression vector. Transfection efficiency of the QMCF/CHO cells was 85%. Cell culture growing details and viability is represented below in Figure 3. To select plasmid-containing cell population, G418 was added 48 h after transfection. 72 h after the 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 the transfection, antibiotic selection was ended and production cell bank was generated (5 vials 1x10^7 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 post transfection G418  was added.
72h post transfection expressoion analysis (WB) was performed.
10 days post transfection cell bank was generated.
Days 11-19 the production phase was performed: temperature was reduced to 30°C, additional nutrients were added to the medium. The viability of the cell culture > 90% was monitored. If viability declined (day 18), the supernatant was clarified and stored at -20°C prior to purification.


Purification and product analysis

Two-step ion-exchange chromatography and gel filtration was used for purification of hMANF. After purification, protein concentration was measured by BCA Protein Assay Kit (Thermo Scientific). BSA (Pierce) was used as a standard. Purity of the protein was analyzed by coomassie-stained SDS-PAGE electrophoresis (Figure 5) and mass-spectrometry (Figure 6).


Figure 5. SDS-PAGE analysis of purified human MANF
Line 1. Protein size marker (PageRuler Prestained protein ladder, Fermentas);
Line 2. 10 µg of purified human MANF.
For visuaization ageBlue protein staining solution (Fermentas) has been used.


Figure 6. Mass-spectometry analysis of purified human MANF. Mass-spectrometry analysis reveal purified hMANF as pure and homogenous material.


Testing of hMANF biological activity

The biological activity of 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.


References

  1. 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
  2. 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)
  3. 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.