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.
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.
Rajan
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Division of Life Sciences, Nelson Laboratories, Rutgers University, Piscataway, New Jersey 08854-8082, USA.
Lackland
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Stein
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Denhardt
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D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017364:Collagenases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D051379:Mice; D010455:Peptides / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry* / Q000235:genetics; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism
D010455:Peptides; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; C033223:polyhistidine; D006639:Histidine; D017364:Collagenases