Highly efficient one-step scarless protein tagging by type IIS restriction endonuclease-mediated precision cloning

Abstract

Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs.

Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases

“…In our recent study to add 11 different tags to the N-terminus of THSD1, a single-span transmembrane protein responsible for cerebral aneurysm pathogenesis (Santiago-Sim et al , 2016), we developed a new cloning strategy by combinational use of type II and type IIS restriction endonucleases (Xu et al , 2017). Unlike type II, type IIS restriction endonucleases recognize non-palindromic sequences and cleave the DNA outside of their recognition site.…”

“…For example, Bsa I recognizes 5′-GGTCTC and cleaves the DNA a nucleotide downstream, resulting in a 5′ overhang 4 nucleotides long, thus making a custom sticky end that matches the gene of interest. Therefore, we can generate a seamless clone by completely eliminating the unwanted junction sequences (Xu et al , 2017). Even more importantly, using type II and type IIS restriction endonucleases in combination makes our method highly compatible with the traditional cloning system that is still widely used by many research labs.…”

“…Traditional DNA recombinant technology based on type II restriction endonucleases renders protein tagging tedious and inefficient as well as the introduction of an unwanted junction sequence. In our attempt to tag Thrombospondin type 1 domain-containing 1 (THSD1) that we identified as the first intracranial aneurysm gene (Santiago-Sim et al , 2016), we developed a novel precision tagging technique by combinational use of type II and IIS restriction endonucleases (Xu et al , 2017), which generates a seamless clone with high efficiency. Here, we describe a protocol that not only provides a generalized strategy for any gene of interest but also takes its application of 11 different tags in THSD1 as a step-by-step example.…”

The Intracranial Aneurysm Gene THSD1 Connects Endosome Dynamics to Nascent Focal Adhesion Assembly

“…Therefore, we hypothesized that THSD1 may be a novel component of FA, a complex macromolecule structure that mediates integrin activation, downstream signaling, and actin cytoskeleton organization [3]. Since commercial antibodies qualified for immunostaining of endogenous THSD1 are not available, we generated a fusion construct that expresses GFP fusion at the C-terminal of THSD1 by our newly described precision tagging technique [9] and confirmed its expression by immunoblotting (Fig. 1F).…”

“…pCMV5-THSD1-GFP and pCMV5-THSD1-FLAG were cloned by a newly-developed precision tagging technique [9]. Wild-type THSD1 or its variants including R450X, R460W, E466G G600E, P639L, T653I, and S775P were amplified by PCR from previous plasmids [2] and sub-cloned into pCMV5 vector.…”