Presence of an N-terminal polyhistidine tag facilitates stable expression of an otherwise unstable N-terminal domain of mouse tissue inhibitor of metalloproteinase-1 in Escherichia coli

Protein Expression and Purification
S S RajanD T Denhardt

Abstract

The active N-terminal domain of the mouse tissue inhibitor of metalloproteinases-1 is a 14.1-kDa polypeptide with three disulfide bonds. When expressed using a T7 system in Escherichia coli, this truncated protein, in contrast to the WT protein, was found only in trace amounts in the cell. However, when the coding sequence was placed downstream of a 60-bp sequence that encoded an in-frame histidine-rich "tag," the fusion product (NF.TIMP*His) was expressed in considerably increased abundance. WT.TIMP-1 was expressed in abundance with or without the tag. The mRNAs encoding the various forms of TIMP were present in similar amounts in all four cases. NF.TIMP*His, renatured and purified on a nickel affinity column, was found to be about 10-fold less effective than native human TIMP-2 at inhibiting cleavage of collagen type I by human fibroblast collagenase. A thrombin cleavage site in the tag was susceptible to cleavage by low levels of a contaminating proteinase.

References

Aug 3, 1992·FEBS Letters·E T CocuzziD T Denhardt
Dec 1, 1993·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·W G Stetler-StevensonD E Kleiner
Jan 1, 1993·Critical Reviews in Oral Biology and Medicine : an Official Publication of the American Association of Oral Biologists·H Birkedal-HansenJ A Engler
Nov 29, 1996·The Journal of Biological Chemistry·J GreeneY E Shi
Nov 5, 1997·Protein Expression and Purification·J NilssonP A Nygren
Feb 3, 1998·Biochemical and Biophysical Research Communications·S S RajanR D Ludescher

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Citations

Mar 31, 2021·Biomacromolecules·Keiichi YoshimatsuKenneth J Shea
Feb 3, 1998·Biochemical and Biophysical Research Communications·S S RajanR D Ludescher
Jan 24, 2006·Protein Expression and Purification·José ArnauJohn Pedersen

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