title: Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein.
abstract: Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro-chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin-resistant under the acidic cleavage conditions (pH 4.5) used in this system. Here, we have modified the pro-chymosin fusion tag and demonstrated that chymosin can remove this tag at more neutral pH (pH 6.2); conditions, that are less prone to compromise the integrity of target proteins. Chymosin was successfully used to produce intact native target protein both at the level of small and large-scale preparations. Using short peptide substrates, we further examined the influence of P1' amino acid (the N-terminus of the native target protein) and found that chymosin accepts many different, although not all, amino acids. We conclude that chymosin has several appealing characteristics for the exact removal of fusion tags. It is readily available in highly purified recombinant versions approved by the FDA for preparation of food for human consumption. We suggest that one should consider extending the use of chymosin to the preparation of pharmaceutical proteins.
authors.affiliation: Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, N Denmark.
authors.forename: Sune F L
authors.initials: SF
authors.lastname: Justesen
authors.forename: Kasper
authors.initials: K
authors.lastname: Lamberth
authors.forename: Lise-Lotte B
authors.initials: LL
authors.lastname: Nielsen
authors.forename: Claus
authors.initials: C
authors.lastname: Schafer-Nielsen
authors.forename: Søren
authors.initials: S
authors.lastname: Buus
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015235:HLA-B Antigens / Q000378:metabolism; D015793:HLA-B7 Antigen; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D015235:HLA-B Antigens; C477444:HLA-B*07:02 antigen; D015793:HLA-B7 Antigen; D011993:Recombinant Fusion Proteins; C032443:prorennin; D012085:Chymosin