Cleavage of different chimeric proteins after specific linker (Asp)4Lys by the highly purified enteropeptidase was investigated, proteins being were accumulated in inclusion bodies or secreted from the cell. Kinetic constants for enzymatic hydrolysis were obtained, indicating that the substrate binding depended mainly on the affinity to the linker peptide (Asp)4Lys. Conditions for the efficient cleavage of recombinant proteins with enteropeptidase are formulated.