Antibody generation within the Human Protein Atlas (HPA) project
The Human Protein Atlas is a Swedish based program initiated in 2003 with the aim to map all the human proteins in cells, tissues and organs using integration of various omics technologies, including antibody-based imaging, mass spectrometry-based proteomics, transcriptomics and systems biology. Our group is responsible for generating the antibodies used in the project.
Since the beginning of the HPA project approximately 57.000 gene fragments (PrESTs, Protein Epitope Signature Tags) have been designed and amplified from a total RNA template pool. Each gene fragment has then been ligated into the expression vector and at the same time fused to an N-terminal hexahistidine tag and an albumin binding protein (His6ABP).
After sequence verification the purified plasmids are transformed into E.coli Rosetta(DE3) cells for production of His6ABP-tagged PrEST-proteins. One-step purification is enabled by the His6-tag and immobilized metal affinity chromatography (IMAC) and performed under denaturing conditions. The purified proteins are then used to prepare antigens for immunization purposes and affinity columns with PrEST-ligands.
Polyclonal antisera generated by the immunization of rabbits (done together with collaborative partners) are carefully purified in a three-step fashion consisting of: depletion of unwanted specificity, capture of wanted specificity (using the affinity columns) and a final buffer exchange step. The binding specificity of all antibodies is determined on protein microarrays to certify that only antibodies with high specificity and low background binding are approved for immunohistochemistry and immunofluorescence analysis. All approved antibodies are further analyzed in a high-throughput Western Blot platform using protein lysates from human cell lines (RT-4 and U-251 MG), human plasma depleted of IgG and HSA and whole tissue lysates from human liver and tonsil. The antibodies will score as Supported or Uncertain depending on the WB results.
