Sortase is one of the most widely used enzymes for covalent protein conjugation that links protein and protein/small molecules together in a site-specific way. It typically recognizes the "GGG" and "LPXTG" peptide sequences and conjugates them into an "LPXTGGG" linker. As a non-natural linker with several flexible glycine residues, it is unknown whether it affects the properties of the conjugated protein. To verify the use of sortase for protein-protein conjugation, we combined single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to characterize sortase-conjugated polyprotein I27 with three different linkers. We found that the I27 with classic linkers "LPETGGG" and "LPETG" from sortase ligation were of normal stability. However, a protein with a longer artificial linker "LPETGGGG" showed a 15% lower unfolding force. MD simulations revealed that the 4G linker showed a high probability of a closed conformation, in which the adjacent monomer has transient protein-protein interaction. Thus, we verify the use of sortase for protein conjugation, and a longer linker with a higher glycine content should be used with caution.