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Star Republic: Guide for Biologists

Purification of inclusion body proteins

Virtualab Protocols (Chang Bioscience)
1. Inclusion body is precipitated, insoluble proteins in bacteria.

2. Make sure your protein is in inclusion body. Membrane-bound protein may also be insoluble without detergent, but can be solublized by including low concentration (<1%) of SDS or Triton X-100.

3. Attempts to avoid inclusion body include growing bacteria at different temperature and using different bacteria strains and culture media.

4. Inclusion bodies are frequently solublized with a buffer that contains 8 M urea or 6 M guanidine hydrochloride.

5. Refolding may be achieved by:

    -Quick dilution, e.g., dilution of the proteins in 8 M urea to 0.4 M urea.
    -Refold on column, e.g., changing buffer from high urea or guanidine hydrochloride to no urea or guanidine hydrochloride while protein is being purified by ion exchange column or affinity column.
    -Slow dialysis against a buffer without denaturant.
    -Refolding in a refolding buffer.