NDLI: Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein.

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Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein.

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Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Lamberth, Kasper Nielsen, Lise-Lotte B. Justesen, Sune F. L. Schafer-Nielsen, Claus Buus, Søren
Description	Author Affiliation: Justesen SF ( Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, N Denmark.)
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.
ISSN	09618368
e-ISSN	1469896X
DOI	10.1002/pro.112
Journal	Protein Science
Issue Number	5
Volume Number	18
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	2009-05-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Chymosin Metabolism Enzyme Precursors Recombinant Fusion Proteins Amino Acid Sequence Animals Genetics Cloning, Molecular Escherichia Coli Hla-b Antigens Hla-b7 Antigen Hydrogen-ion Concentration Mass Spectrometry Molecular Sequence Data Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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