Structure of folding intermediates at pH 4.0 and native state of microbial transglutaminase.
Biosci Biotechnol Biochem
; 67(2): 291-4, 2003 Feb.
Article
in En
| MEDLINE
| ID: mdl-12728988
ABSTRACT
Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.0 to 6.0. CD analysis showed that a burst of secondary structure formation occurred within the dead time of the experiment and accounted for 75% of the signal change in the far UV CD, with little tertiary structure being formed. This burst was followed by slow rearrangement of the secondary structure accompanied by formation of tertiary structure. The secondary and tertiary structures of the final sample at pH 4.0, corresponding to the folding intermediate, were different from these structures at pH 6.0. Once the native structure was obtained, acidification of the native protein to pH 4.0 did not lead to a structure like that of the folding intermediate. Sedimentation velocity analysis showed that the folding intermediate had an expanded structure and contained no other structure species including large aggregates.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Streptomycetaceae
/
Transglutaminases
Language:
En
Journal:
Biosci Biotechnol Biochem
Journal subject:
BIOQUIMICA
/
BIOTECNOLOGIA
Year:
2003
Document type:
Article
Affiliation country: