Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases.
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.
Kühnel
Blanka
B
SemBioSys Genetics Inc., 110 2985 23rd Avenue NE, Calgary AB T1Y 7L3, Canada.
Alcantara
Joenel
J
Boothe
Joseph
J
van Rooijen
Gijs
G
Moloney
Maurice
M
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D012085:Chymosin / Q000378:metabolism; D006629:Hirudins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011498:Protein Precursors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
D006629:Hirudins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013917:Thrombin; D016282:Aspartic Acid Endopeptidases; D012085:Chymosin