Generation of Proteins with Free N-Terminal Cysteine by Aminopeptidases

Abstract

Efficient, site-specific, and bio-orthogonal conjugation of chemical functionalities to proteins is of great utility in fundamental research as well as industrial processes (e.g., the production of antibody–drug conjugates and immobilization of enzymes for biocatalysis). A popular approach involves reacting a free N-terminal cysteine with a variety of electrophilic reagents. However, current methods for generating proteins with N-terminal cysteines have significant limitations. Herein we report a novel, efficient, and convenient method for producing recombinant proteins with free N-terminal cysteines by genetically fusing a Met-Pro-Cys sequence to the N-terminus of a protein of interest and subjecting the recombinant protein to the sequential action of methionine and proline aminopeptidases. The resulting protein was site-specifically labeled at the N-terminus with fluorescein and a cyclic cell-penetrating peptide through native chemical ligation and a 2-cyanobenzothiazole moiety, respectively. In addition, the optimal recognition sequence of Aeromonas sobria proline aminopeptidase was determined by screening a combinatorial peptide library and incorporated into the N-terminus of a protein of interest for most efficient N-terminal processing.

Catalyst-free thiazolidine formation chemistry enables the facile construction of peptide/protein–cell conjugates (PCCs) at physiological pH

“…44,45 Although several approaches have been developed to produce proteins with an N-terminal free Cys, it remains a labor-intensive task involving enzyme-mediated cleavage and subsequent stepwise protein purification procedures. 46,47 For ease of manipulation, we decided to develop a SpyCatcher–SpyTag Chemistry-Assisted Cell Surface Engineering (SpyCASE) strategy through the combined use of thiazolidine formation and the SpyCatcher–SpyTag system (Fig. 2A).…”

Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine

“…Alternatively, Met-Cys is expressed at the N-terminus, which is recognized by endogenous methionine aminopeptidases that cleave the Met residue and expose the 1,2-aminothiol motif . Recently, a more efficient and highly specific approach was developed in which a Met-Pro-Cys sequence with a short peptidase recognition motif (4–10 residues) is attached to the protein N-terminus and subjected to recombinant methionine and proline aminopeptidases . In addition, 1,2-aminothiols can be introduced into other sites in the protein sequence by using unnatural amino acids containing this particular substructure …”

“…4 Recently, a more efficient and highly specific approach was developed in which a Met-Pro-Cys sequence with a short peptidase recognition motif (4−10 residues) is attached to the protein N-terminus and subjected to recombinant methionine and proline aminopeptidases. 1 In addition, 1,2-aminothiols can be introduced into other sites in the protein sequence by using unnatural amino acids containing this particular substructure. 5 Several electrophilic groups were developed to selectively target proteins with N-terminal Cys, 6−10 e.g., thioesters, 11 Osalicylaldehyde esters, 12 activated aldehydes, 8 activated nitriles, 13 2-((alkylthio)(aryl)methylene)malononitriles, 14 Nhydroxysuccinimide-activated acrylamides, 15 2-benzylacrylaldehydes, 16 2-formylphenylboronic acids, 17−19 and monosubstituted cyclopropenones.…”

“…The 1,2-aminothiol moiety is rarely found in nature, yet its unique property, i.e., binucleophilicity, makes it a useful functionality for manipulating molecules in complex matrices such as intracellular environments. For example, proteins containing N-terminal cysteine (Cys) can be site-specifically modified at this solvent-exposed site, and the modifications have minimal structural and functional effects . Although such proteins are rare in nature, this small tag can be readily engineered into any protein.…”

“…For example, proteins containing N-terminal cysteine (Cys) can be site-specifically modified at this solvent-exposed site, and the modifications have minimal structural and functional effects. 1 Although such proteins are rare in nature, this small tag can be readily engineered into any protein. Most commonly, proteins are expressed with a recognition sequence for a sequence-selective protease (e.g., tobacco etch virus, thrombin, or factor Xa), 2,3 and the selective cleavage of the tag yields a protein with free N-terminal Cys.…”