The present invention relates to a process of separation a protein of interest comprising expressing the protein of interest fused with a fusion partner, selectively adsorbing on matrix, and performing efficient cleavage reaction on the fusion protein adsorbed on matrix or separated from the matrix, and recovering the protein of interest. Also, the present invention provides a fusion protein comprising a protein of interest and a fusion partner, wherein the fusion partner comprises an amino acid sequence which can be cleaved by ubiquitin cleavage enzyme at its C-terminus, and which has a difference in isoelectric point of 1 or more from the protein of interest. According to the present invention, the proteins of interest can be purified in high yield and purity. In addition, complicated separating processes required in the production of recombinant proteins can be eliminated so as to cut down the production cost.