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Protein Engineering vol. 16 no. 10 pp. 777-783, 2003
© 2003 Oxford University Press

Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases

Blanka Kühnel1, Joenel Alcantara2, Joseph Boothe1, Gijs van Rooijen3 and Maurice Moloney1,2,4

1SemBioSys Genetics Inc., 110 2985 23rd Avenue NE, Calgary AB T1Y 7L3, 2Department of Biosciences, University of Calgary, 2500 University Drive NW, Calgary AB T2N 1N4 and 3GenomePrairie, 115 3553 31st Street NW, Calgary AB T2L 2K7, Canada

4 To whom correspondence should be addressed. e-mail: moloneym{at}sembiosys.com

Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. In general, such fusion proteins must be cleaved to release the mature protein in its native form. The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity. We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin. DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host. Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein. The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds. Even when the fusion protein was sequestered and immobilized on oilbodies, precise and efficient cleavage was obtained. The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.

Received June 5, 2003; revised August 1, 2003; accepted August 20, 2003.


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