School of Biotechnology

10 selected innovations and scientific achievements
(Uhlen and co-workers)

Affinity tag concept (1983)
The first affinity tag for purification of recombinant proteins was based on protein A1. In the following years, applications for immobilization2 and antibody generation3 were described. The use of dual affinity tags was also developed4 to take advantage of two affinity events for purification of recombinant proteins. The use of affinity tags has since become wide-spread as a versatile tool in bioscience5.

Protein A and G for antibody purification (1984)

The genes for protein A6 and protein G7 were cloned and characterized and several recombinant variants of these bacterial immunoglobulin-binding receptors were developed8. Recombinant protein A and G are now frequently used for affinity purification of antibodies to be used as research reagents, diagnostics or therapeutics.
Solid phase sequencing (1988)
The principle of solid phase DNA sequencing was described based on binding of biotinylated DNA to streptavidin coated magnetic beads and elution of one strand selectively using alkali9. The method allowed robotic applications10 suitable for clinical sequencing11, but the magnetic handling has also found frequent use in many molecular applications, including sample handling for DNA diagnostics12. The use of solid phase methods is now frequently used as an integrated part of many of the next generation DNA sequencing methods.
Extended half life of recombinant proteins (1991)
A new principle for increasing the in vivo half-life of therapeutic proteins was described13. The method is based on serum albumin binding domain (ABD) as fusion partner for proteins of therapeutic value. Recently, engineered versions of this domain have been selected with sub-picomolar affinity to human serum albumin.
Gram positive bacterial display (1992)
A new approach for surface display of proteins was described based on the Gram positive bacterium14. The surface display have been used for subunit vaccine delivery, whole cell tools for diagnostic and environmental applications15. Recently, improvements have been made with results indicate that bacterial surface display of protein libraries combined with FACS sorting might indeed constitute an attractive alternative to existing technology platforms for high-throughput affinity based selections.
Pyrosequencing (1993)
A new principle, Pyrosequencing, for polymerase-based DNA sequencing by synthesis was developed taking advantage of the detection of pyrophosphate release through a combination of enzymes to generate light. Solid phase methods, based on binding to streptavidin coated beads, were developed for mini-sequencing16 and DNA sequencing17. A solution-based method was subsequently described18 in which the enzyme apyrase was used to remove non-incorporated nucleotides. Recently, a “next generation DNA sequencing” instrument was released (454/Roche) based on the solid phase version of pyrosequencing, in combination with microfluidics to allow parallel analysis of many samples.
Affibodies (1995)
A new scaffold19 for generation of protein binders called affibodies based on combinatorial protein engineering of a small three-helix bundle was described20. Affibodies have been used as selective binding reagents in many biotechnology and biomedical applications, including targeting of tumor antigens for imaging and therapy and affinity reagents for separation technology21. The use of scaffolds as an alternative to antibodies has increased substantially during the last years.
Stabilization of proteins to high pH (2000)
Most protein-based affinity chromatography media show significant fragility towards alkaline conditions. A new approach to generate protein stable at alkaline conditions was developed based on protein engineering of aspargine residues22. Recently, a new affinity matrix MabSelectSure for affinity capture of antibodies were released (GE Health, Uppsala), based on this new engineering approach, allowing cleaning-in-place using 0,1 – 0,5 M NaOH.
A human protein atlas (2005)
An antibody-based Human Protein Atlas has been created ( to show the expression and localization of proteins in a large variety of normal human tissues, cancer cells and cell lines23. The data is presented as high-resolution images with immunohistochemically stained tissue sections and immunofluorescent24 labeled cell lines. In August 2008, version 4 of the protein atlas was launched25 based 5,000 proteins corresponding to 25% of the human protein-encoding genes.
Epitope mapping using bacterial display (2008)
A novel method for epitope mapping of antibodies based on bacterial surface expression of protein fragments followed by antibody-based flow-cytometric sorting was described26. Using the approach, bacterial cells with displayed protein fragments can also be used as affinity ligands to generate epitope-specific antibodies. The results show a path forward for systematic validation of antibodies with regards to epitope specificity and cross-reactivity on a whole proteome level.
Selected references
Uhlen, Nilsson, Guss, Lindberg, Gatenbeck, and Philipson (1983) “Gene fusion vectors based on the gene for staphylococcal protein A” Gene 23, 369-378.
Nilsson, Abrahmsen, and Uhlen (1985) “Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors” EMBO J 4, 1075-1080.
Lowenadler, Nilsson, Abrahmsen, Moks, Ljungqvist, Holmgren, Paleus, Josephson, Philipson, and Uhlen (1986) “Production of specific antibodies against protein A fusion proteins” EMBO J 5, 2393-2398.
Hammarberg, Nygren, Holmgren, Elmblad, Tally, Hellman, Moks and Uhlen (1989) “Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II” Proc Natl Acad Sci
, 86(12): p. 4367-71
Uhlen (2008) “Affinity as a tool in life science” Biotechniques 44: S649-S654
Uhlen, Guss, Nilsson, Gatenbeck, Philipson, and Lindberg (1984) “Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications” J Biol Chem 259, 1695-1702.
Guss, Eliasson, Olsson, Uhlen, Frej, Jornvall, Flock, and Lindberg (1986) “Structure of the IgG-binding regions of streptococcal protein G” EMBO J 5, 1567-1575.
Nilsson, Moks, Jansson, Abrahmsen, Elmblad, Holmgren, Henrichson, Jones, and Uhlen (1987) “A synthetic IgG-binding domain based on staphylococcal protein A” Protein Eng 1, 107-113.
Stahl, Hultman, Olsson, Moks and Uhlen (1988) “Solid phase DNA sequencing using the biotin-avidin system” Nucleic Acids Res 16, 3025-3038.
Hultman, Stahl, Hornes and Uhlen (1989) “Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support” Nucleic Acids Res 17, 4937-4946.
Leitner, Escanilla, Franzen, Uhlen and Albert (1996) “Accurate reconstruction of a known HIV-1 transmission history by phylogenetic tree analysis” Proc Natl Acad Sci U S A 93, 10864-10869.
Uhlen (1989) “Magnetic separation of DNA” Nature 340, 733-734.
Nygren, Flodby, Andersson, Wigzell, and Uhlén [1991] “In vivo stabilization of a human recombinant CD4 derivative by fusion to a serum-albumin-binding receptor” Vaccines 91, Cold Spring Harbor Laboratory. 363-368.
Hansson, Ståhl, Nguyen, Bächi, Robert, Binz, Sjölander, and Uhlén (1992) “Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus” J. Bacteriol. 174: 4239-4245.
Ståhl, and Uhlén (1997) “Bacterial surface display: trends and progress” Trends Biotechnology 15: 185-192.
Nyren, Pettersson and Uhlen (1993) “Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay” Anal Biochem 208, 171-175.
Ronaghi, Karamohamed, Pettersson, Uhlen and Nyren (1996) “Real-time DNA sequencing using detection of pyrophosphate release” Anal Biochem 242, 84-89.
Ronaghi, Uhlen and Nyren (1998) “A sequencing method based on real-time pyrophosphate” Science 281, 363-365.
Nord, Nilsson, Nilsson, Uhlen and Nygren (1995) “A combinatorial library of an alpha-helical bacterial receptor domain” Protein Eng 8, 601-608.
Nord et al. (1996) Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nature Biotechnology 15, 772-777.
Nygren and Uhlen (1997) “Scaffolds for engineering novel binding sites in proteins” Curr Opin Struct Biol 7, 463-469.
Gülich, Linhult, Uhlén, Nygren and Hober S. (2000) “Stability towards alkaline conditions can be engineered into a protein ligand” J. Biotechn., 80, 169-178.
Uhlen et al (2005) “A human protein atlas for normal and cancer tissues based on antibody proteomics” Mol Cell Proteomics 4(12):1920-1932
Barbe, Lundberg, Oksvold, Stenius, Lewin, Björling, Asplund, Pontén, Brismar, Uhlén and Andersson-Svahn (2008) “Toward a confocal subcellular atlas of the human proteome” Mol Cell Proteomics 7(3):499-508.
Berglund, Björling, Oksvold, Fagerberg, Asplund, Szigyarto, Persson, Ottosson, Wernérus, Nilsson, Lundberg, Sivertsson, Navani, Wester Kampf. Hober, Pontén and Uhlen (2008) “A gene-centric human protein atlas for expression profiles based on antibodies” Mol Cell Proteomics 7(10):2019-27.
Rockberg. Löfblom, Hjelm, Uhlen and Ståhl (2008) “Epitope mapping of antibodies using bacterial surface display” Nature Methods, in press.
Last updated: 2010-12-22