An Atypical Naturally Split Intein Engineered for Highly Efficient Protein Labeling

Abstract

Protein trans‐splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL‐TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N‐terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans‐splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50‐fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi‐synthesis and other intein‐related biotechnological applications.

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

“…Fig. 8 shows that the AceL-TerL intein was fully active in the trans-splicing assay using pep-C1, as reported previously (25), however, no splice product formation or cleavage reaction could be observed with pep-C1S. Thus, the AceLTerL intein does not support protein splicing with the Ser1/ Cysϩ1 combination.…”

“…The small size of this fragment of 37 or 28 aa also calls for applications using semisynthetic protein trans-splicing (23,25,51,52).…”

“…By using the amino acid sequence of the AceL-TerL intein (25) as search query this intein was found in the metagenomic database at NCBI. The two fragments were identified manually and the putative intein boundaries were defined by the low homology to other inteins.…”

“…According to its origin from Punta Cormorant in the global ocean sampling project and its integration point in a terminase large subunit, we referred to the intein as GOS-TerL. The closest homolog of the GOS-TerL intein is the very recently reported naturally split AceL-TerL intein (129 aa) (25) (Fig. 2).…”

“…Very recently, the first atypically split intein was discovered in metagenomic databases and biochemically characterized. The AceL-TerL intein consists of a short Int N fragment of 25 aa and a C-terminal fragment of 104 aa (25). Metagenomic databases proved already earlier to be a rich source for new split inteins with interesting properties (26,27).…”