Skip to main content
SpringerLink
Log in

High speed immuno-affinity chromatography on supports with gigapores and porous glass

  • Published:
Bioseparation

Abstract

Immuno-affinity chromatography exploiting the Ca2+ dependent interaction of the anti-Flag antibody and Flag-tagged proteins has been investigated. The antibody has been immobilized on porous glass beads (Prosep) containing gigapores and on a monolith, the polymethacrylate based Convective Interactive Media (CIM) column at a ligand density of 2 mg/g and 10 mg/ml respectively. The performance of the columns was assessed by applying clarified yeast culture supernatant containing overexpressed Flag-human serum albumin. Dynamic binding capacity and purity was checked at various flow rates ranging from 100 cm/h to 800 cm/h. 95% purity could be obtained. Anti Flag-CIM columns showed a higher unspecific adsorption, requiring a longer wash cycle to obtain the same purity compared to the Prosep column. Anti Flag-CIM columns showed a flow independent performance, which is explained by its monolithic structure. A decreasing dynamic binding capacity with flow was observed with anti-Flag-Prosep columns. Both columns are suited to purify milligrams of protein out of a yeast culture supernatant within a few minutes. We considered them as promising candidates for high throughput screening, where fast purification is a necessity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Abou-Rebyeh H, Körber F, Schubert-Rehberg K, Reusch J and Josic D (1991) Carrier membrane as a stationary phase for affinity chromatography and kinetic studies of membrane-bound enzymes. J. Chromatogr. 566: 341–350.

    Google Scholar 

  • Amatschek K, Necina R, Hahn R, Schallaun E, Schwinn H, Josic D and Jungbauer A (2000) Affinity chromatography of human blood coagulation factor VIII on monoliths with peptides from a combinatorial library. J. High Res. Chromatogr. 23: 47–58.

    Google Scholar 

  • Babashak JV and Phillips TM (1989) Isolation of a specific membrane protein by immuno-affinity chromatography with biotinylated antibodies immobilized on avidin-coated glass beads. J. Chromatogr. 476: 187–194.

    Google Scholar 

  • Bjerrum OJ and Heegard NHH (1988) Handbook of immunoblotting of Proteins. CRC Press, Boca Raton, FL.

    Google Scholar 

  • Brake AJ (1989) Secretion of heterologous proteins directed by the yeast alpha-factor leader. Bio/Technology 13: 269–280.

    Google Scholar 

  • Brake AJ (1990) Alpha-factor leader-directed secretion of heterologous proteins from yeast. Methods Enzymol. 185: 408–421.

    Google Scholar 

  • Brake AJ, Merryweather JP, Coit DG, Heberlein UA, Masiarz FR, Mullenbach GT, Urdea MS, Valenzuela P and Barr PJ (1984) Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81: 4642–4646.

    Google Scholar 

  • Buckholz RG and Gleeson MAG (1992) Foreign gene expression in yeast: a review. Bio/Technology 9: 1067–1072.

    Google Scholar 

  • Ford CF, Suominen I and Glatz CE (1991) Fusion tails for the recovery and purification of recombinant proteins. Protein Expr. Purif. 2: 95–107.

    Google Scholar 

  • Füglistaller P (1989) Comparison of immunoglobulin binding capacities and ligand leakage using eight different protein A affinity chromatography matrices. J. Immunol. Methods 124: 171–177.

    Google Scholar 

  • Gellissen G, Melber K, Janowicz ZA, Dahlems UM, Weydemann U, Piontek M, Strasser AW and Hollenberg CP (1992) Heterologous protein production in yeast. Antonie Van Leeuwenhoek 62: 79–93.

    Google Scholar 

  • Hagedorn J, Kaspar C, Freitag R and Tennikova T (1999) High performance flow injection analysis of recombinant protein G. J. Biotechnol. 69: 1–7.

    Google Scholar 

  • Hahn R, Amatschek K, Schallaun, Necina R, Josic D and Jungbauer A (2000) Performance of affinity chromatography with peptide ligands: influence of spacer, matrix composition, and immobilization chemistry. Int. J. Biochromatogr. (in press).

  • Heeter GA and Liapis A 1(1997) Model discrimination and estimation of the intraparticle mass transfer parameters for the adsorption of bovine serum albumin onto porous adsorbent particles by the use of experimental frontal analysis data. J. Chromatogr. A 776: 3–13.

    Google Scholar 

  • Heukeshoven J and Dernick R (1988) Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis 9: 28–32.

    Google Scholar 

  • Hopp TP, Prickett KS, Price V, Libby RT, March CJ, Cerretti P, Urdal DL and Conlon P J (1988) A short polypeptide marker squence useful for recombinant protein identification and purification. Bio/Technology 6: 1205–1210.

    Google Scholar 

  • Iberer G, Hahn R and Jungbauer A (1999) Monoliths as stationary phases for separating biopolymers. LC-GC 17: 998–1005.

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Google Scholar 

  • Maier E, Meier-Ewert S, Ahmadi AR, Curtis J and Lehrach H (1994) Application of robotic technology to automated sequence fingerprint analysis by oligonucleotide hybridisation. J. Biotechnol. 35: 191–203.

    Google Scholar 

  • Nilsson J, Nilsson P, Williams Y, Pettersson L, Uhlén M and Nygren PA (1994) Competitive elution of protein A fusion proteins allows specific recovery under mild conditions. Eur. J. Biochem. 224: 103–108.

    Google Scholar 

  • Nilsson J, Ståhl S, Lundeberg J, Uhlén M and Nygren PA (1997) Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11: 1–16.

    Google Scholar 

  • Petropoulos JH, Liapis AI, Kolliopoulos NP, Petrou JK and Kanellopoulos NK (1990) Restricted diffusion of molecules in porous affinity chromatography adsorbents. Bioseparation 1: 69–88.

    Google Scholar 

  • Platanova GA, Pankova GA, Il'ina IY, Vlasov GP and Tenikova TB (1999) Qualitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography. J. Chromatogr. A 852: 129–140.

    Google Scholar 

  • Podgornik H, Podgornik A and Perdih A (1999) A method of fast separation of lignin peroxidases using convective interaction media disks. Anal. Biochem. 272: 43–47.

    Google Scholar 

  • Podgornik A, Barut M, Strancar A, Josic D and Koloini T (2000) Construction of large volume monolithic columns. Anal. Chem. 72: 5693–5699.

    Google Scholar 

  • Prickett KS, Amberg DC and Hopp TP (1989) A calcium dependent antibody for identification and purification of recombinant proteins. Bio Techniques 7: 580–589.

    Google Scholar 

  • Schuster M, Wasserbauer E, Ortner C, Graumann K, Jungbauer A, Hammerschmid F and Werner G (2000) Shortcut of protein purification by integration of cell disrupture and affinity extraction. Bioseparation 9: 59–67.

    Google Scholar 

  • Strancar A and Koselj P (1996) Application of compact porous disks for fast separations of biopolymers and in-process control in biotechnology. Anal. Chem. 68: 3483–3488.

    Google Scholar 

  • Strancar A, Barut M, Podgornik A, Koselj P, Schwmnn H, Raspor P and Josic D (1997) Application of compact porous tubes for preparative isolation of clotting factor VIII from human plasma. J. Chromatogr. A 760: 117–123.

    Google Scholar 

  • Strancar A, Barut M, Podgornik A, Koselj P, Josic D and Buchacher A (1998) Convective interaction media: polymer-based supports for fast separation of biomolecules. LC-GC 11: 660–670.

    Google Scholar 

  • Swaisgood HE and Chaiken IM (1985) Quantitative affinity high-performance liquid chromatography of neuro endocrine polypeptides using porous and non-porous glass derivatives. J. Chromatogr. 327: 193–204.

    Google Scholar 

  • Tennikov MB, Gazdina NV, Tennikova TB and Svec F (1998) Effect of porous structure of macroporous polymer supports on resolution in high-performance membrane chromatography of proteins. J. Chromatogr. A 798: 55–64.

    Google Scholar 

  • Tennikova TB and Svec F (1993) High-performance membrane chromatography: highly efficient separation method for proteins in ion-exchange, hydrophobic interaction and reversed phase modes. J. Chromatogr. 646: 279–288.

    Google Scholar 

  • Tennikova T, Svec F and Belenkii B G (1990) High-performance membrane chromatography. A novel method of protein separation. J. Liq. Chromatogr. 13: 63.

    Google Scholar 

  • Tennikova TB, Bleha M, Svec F, Almazova TV, and Belenkii BG (1991) High-performance membrane chromatography of proteins, a novel method for protein separation. J Chromatogr 555: 97–107.

    Google Scholar 

  • Tyn MT and Gusek TW (1990) Prediction of diffusion coefficients of proteins. Biotech. Bioeng. 35: 327–338.

    Google Scholar 

  • Widjojoatmodjo MN, Fluit AC, Torensma R and Verhoef J (1993) Comparison of inununomagnetic beads coated with protein A, protein G, or goat anti-mouse inimunoglobulins. Applications in enzyme immunoassays and immunomagnetic separations. J Immunol. Methods 165: 11–19.

    Google Scholar 

Download references

Author information

Author notes

  1. M. Schuster, E. Wasserbauer & A. Neubauer

    Present address: IGENEON GmbH, Brunnerstr. 59, 1235, Vienna, Austria

Authors and Affiliations

  1. Novartis Forschungsinstitut, Brunnerstr. 59, 1235, Vienna, Austria

    M. Schuster, E. Wasserbauer & A. Neubauer

  2. Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, 1190, Vienna, Austria

    M. Schuster & A. Jungbauer

Authors
  1. M. Schuster
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Wasserbauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Neubauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Jungbauer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schuster, M., Wasserbauer, E., Neubauer, A. et al. High speed immuno-affinity chromatography on supports with gigapores and porous glass. Bioseparation 9, 259–268 (2000). https://doi.org/10.1023/A:1011130821804

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1011130821804

Search

Navigation