A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa.
Expression and purification of proteins in recombinant DNA systems is a powerful and widely used technique. Frequently there is the need to express the protein of interest as a fusion protein or chimeric protein. Fusion protein technology is frequently used to attach a "signal" which can be used for subsequent localization of the protein or a "carrier" which can be used to deliver a "therapeutic" such as a radioactive molecule to a specific site. In addition to these applications, fusion protein technology can be employed for several other useful purposes. Of these, the most frequent reason is to provide a 'tag' or 'handle' which will aid in the purification of the protein. Another useful purpose is to improve the expression or folding of the protein of interest. In these latter two situations, it is often necessary to remove the fusion partner before the recombinant protein of interest can be used for further studies. This removal process involves the insertion of a unique amino acid sequence that is susceptible to cleavage by a highly specific protease. Thrombin and factor Xa are the most frequently used proteases for this application. The purpose of this review is to discuss the application of thrombin and factor Xa for the cleavage of fusion proteins. It is emphasized that while these enzymes are quite specific for cleavage at the inserted cleavage site, proteolysis can frequently occur at other site(s) in the protein of interest. It is necessary to characterize the protein of interest after cleavage from the affinity label to assure that there are no changes in the covalent structure of the protein of interest. Examples are presented which describe the proteolysis of the protein of interest by either factor Xa or thrombin.
Jenny
Richard J
RJ
Haematologic Technologies, Inc., Essex Junction, VT, USA. rjenny@haemtech.net
Mann
Kenneth G
KG
Lundblad
Roger L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004765:Enteropeptidase / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006629:Hirudins / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D006801:Humans; D006868:Hydrolysis; D058619:Maltose-Binding Proteins; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D036361:Peptide Hormones / Q000378:metabolism; D018272:Porins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
D002352:Carrier Proteins; D006629:Hirudins; C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; C109237:OmpF protein; D036361:Peptide Hormones; D018272:Porins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D005982:Glutathione Transferase; D010450:Endopeptidases; C099488:TEV protease; D013917:Thrombin; D015951:Factor Xa; D004765:Enteropeptidase