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Related Experiment Videos

Continuous chromatographic protein refolding.

Heikki Lanckriet1, Anton P J Middelberg

  • 1Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, UK.

Journal of Chromatography. A
|February 3, 2004
PubMed
Summary
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Size-exclusion chromatography (SEC) refolding offers advantages over batch dilution for protein refolding, especially regarding denaturant removal. Continuous SEC refolding using preparative continuous annular chromatography (P-CAC) achieves high yields at high protein concentrations.

Area of Science:

  • Biochemistry and Biotechnology
  • Protein Science
  • Chromatographic Techniques

Background:

  • Column-based protein refolding is crucial for producing substantial protein quantities.
  • Size-exclusion chromatography (SEC) refolding separates unfolded proteins from denaturants using specific matrices.
  • Lysozyme is utilized as a model protein to compare refolding methods.

Purpose of the Study:

  • To compare size-exclusion chromatography (SEC) refolding with batch dilution refolding.
  • To evaluate the impact of chemical composition, specifically dithiothreitol (DTT) concentration, on refolding yield.
  • To assess the scalability and efficiency of continuous SEC refolding using preparative continuous annular chromatography (P-CAC).

Main Methods:

  • Comparative analysis of SEC refolding and batch dilution refolding with lysozyme.

Related Experiment Videos

  • Investigation of refolding buffer composition, including dithiothreitol (DTT) concentration.
  • Adaptation of batch SEC refolding to a continuous preparative continuous annular chromatography (P-CAC) system.

    • Main Results:

    • Lysozyme refolding yield is highly sensitive to refolding buffer composition, particularly DTT concentration.
    • SEC refolding is robust against DTT carry-over due to contaminant separation.
    • SEC refolding yields are comparable to batch dilution at low protein concentrations, with no significant advantage at higher concentrations.
    • Continuous P-CAC system demonstrates similar elution profiles to stationary columns, facilitating scale-up.
    • High refolding yields (72%) achieved at high protein concentrations (>1 mg/mL) using P-CAC.

    Conclusions:

    • SEC refolding is less susceptible to buffer contaminants like DTT compared to batch dilution.
    • Continuous preparative continuous annular chromatography (P-CAC) offers a scalable solution for efficient protein refolding at high concentrations.
    • P-CAC enables high refolding yields, crucial for industrial protein production.