Center for Proteomics was founded in 2006 as a core facility for mAb production. The project’s team has supervised the development of mouse mAbs to ≥ 10 antigens per year (> 100 antigens in total). To that aim, we have implemented several protein production lines for immunogen preparation and the laboratories for Ab purification and characterization. Our workflow protocols are fully optimized and supported by acquisition of ISO certificate in 2018 (ISO 9001:2015).
In order to obtain antigens needed for immunization proteins will be expressed using eukaryotic (mammalian) or prokaryotic (bacterial) expression system. Cloning vectors, in other words genetically engineered plasmids encoding each protein/ORF will be ordered.
Membrane and glycosylated proteins will be expressed using eukaryotic expression vector that allows generation of mouse IgG Fc fusion protein. Most of the eukaryotic proteins, especially the ones directed to cell membrane or secretion, undergo post-translational modifications, and one of the most common is attachment of glycans to protein backbone (glycosylation). Although glycosylation exists in prokaryotes as well, the composition of the glycans, their branching and abundance, substantially differ between eukaryotes and prokaryotes. Therefore, in order to mimic natural glycosylation patterns, nucleotide sequence of viral proteins presumed to be glycosylated or localized on the cell membranes will be subcloned into expression plasmid, allowing production of secreted protein in eukaryotic cells coupled with addition of mouse IgG Fc protein tag for purification. In particular, after subcloning and verification of nucleotide sequence insertion, expression plasmid will be used to transfect eukaryotic HEK239T cells resulting in their secretion of the Fc fusion protein. Purification and quantification of this proteins will be performed using HPLC chromatography on AKTA purifier by using protein A or G sepharose columns. Protein stability and purity will be analysed by gel electrophoresis. Purified proteins will be used for immunization of animals.
Other proteins, presumed to be localized in nucleus or cytoplasm, will be expressed in E. coli using prokaryotic expression vector that adds terminal His-tag to proteins. Protein expression in E. coli has been well described in literature and is one of the fastest, most economic and most often used strategy to obtain large protein quantities. Center for Proteomics has years-long experience in production of immunogens by bacterial expression system. In brief, nucleotide sequence of target viral proteins will be subcloned into inducible prokaryotic expression plasmid that allows production of protein in E. coli coupled with addition of HIS tag for purification. After subcloning, these plasmids will be used to transform competent E. coli strain, that will, upon addition of induction reagents IPTG, start producing larger quantities of target protein. Expressed protein will be isolated and purified using AKTA purifier with Ni or Co columns. As for Fc fusion proteins, the protein stability and purity will be analysed by gel electrophoresis.
(Refer to the flowchart to select the best option for your needs. Estimated duration of each step of immunogen generation is shown in the table below.)
1. Production of protein of interest from E.coli or Fc-fusion expression vectors.
Please note that the Center for Proteomics is not cloning custom constructs; we start the protein production from the vector containing your gene construct suitable for E.coli production or production in mammalian cells.
Selecting the proper expression system is not a trivial question and is summarized in the flowchart. Bacterial expression is robust, quick and produces high yields. However, if your protein is heavily glycosylated and/or you wish an antibody that will work against it in its native conformation (for instance in FC or IF), it may not be best suited. Additionally, the desired protein may be toxic for bacteria or may be difficult to purify. For protein expression in E.coli we will accept any construct carrying the gene for His tagged protein or GST tagged protein, and the gene for common antibiotic resistance.
For expression of glycosylated proteins we recommend Fc-fusion protein expression. In addition to being good antigens for immunization, Fc-fusion proteins are a tool on their own and can be used in FACS or IP to look for interaction partners. When making a construct please bear in mind that a) only the ectodomain and not the full protein sequence is cloned and b) the natural signal sequence is not present in the final construct. Otherwise, we accept any IgG Fc fusion vector available on the market. For additional details, please refer to the flowchart below.
We have a good experience with peptide immunization. Please provide us with 0,5 mg of KLH coupled peptide and 0,5 mg of BSA coupled peptide of your interest.
2. Purification of expressed protein using affinity chromatography (please refer to table 2 for details)
Please note that the Center for Proteomics is not cloning custom constructs, we start the protein production from the vector containing your gene construct suitable for E. coli production or production in mammalian cells. We will accept any construct carrying the gene for His tagged protein or the GST tagged protein, and the gene for common antibiotic resistance. We will accept any IgG Fc fusion vector available on the market. We have a good experience with peptide immunization. Please provide us with 0,5 mg of KLH coupled peptide and 0,5 mg of BSA coupled peptide of your interest.
We use at least two animals per immunization and select the best responders using ELISA prior to final boost and fusion. Please refer to the chart below for estimate about the duration of immunization.
Generation of custom hybridoma cell line stably expressing mouse monoclonal antibodies specific to your target protein. Number of different hybridomas generated and scale is flexible and can be tailored to your needs. Please refer to the table below for estimate about the duration of individual steps in generation of hybridomas.
Cell culture, antibody purification and fragmentation:
1. Testing and selection of hybridomas in different applications and techniques (FACS, WB, IP, FC)
2. Isotyping and/or clone confirmation using ELISA (for mouse IgGs)
3. mAb purification using protein A or G affinity chromatography
4. Adaptation of hybridomas to serum-free medium (please note that the standard cell medium contains 10% FBS and bovine IgGs that cannot be separated from your mAbs unless the cells are adapted to the serum free medium)
5. Antibody fragmentation using papain
6. Manipulation with your existing hybridoma cell lines (please note that services 1-5 are possible also for the hybridoma cell line that you already have in your lab, but e.g. you want to be sure in its clonality, or you want to have it adapted to the serum free medium so the mAbs can be purified out of it).
