Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein
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RESEARCH PAPER
Improved protein purification system based on C‑terminal cleavage of Npu DnaE split intein Hai‑Feng Xia1,2 · Ting‑Jun Zhou1 · Ye‑Xing Du1 · Yu‑Jun Wang1 · Chang‑Hua Shi3 · David W. Wood3 Received: 25 February 2020 / Accepted: 17 May 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on I N affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9–21.0 mg mL−1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production. Keywords Protein purification · C-terminal cleavage · Trans-splicing · Npu DnaE intein · Affinity chromatography
Introduction Protein expression and purification is one of the most important research topics today. The use of affinity tags greatly simplifies recombinant protein purification but requires additional time and cost for their removal [1, 2]. The discovery of inteins provides a new solution to this problem. Inteins are internal protein sequences that can excise themselves from premature host proteins and concomitantly ligate the flanking sequences by a native peptide bond, called protein splicing [3–6]. The mechanism of protein splicing includes four nucleophilic reaction steps: (1) a N-S or N-O acyl shift, (2) transesterification, (3) Asn cyclization and (4) an X-N acyl shift [7]. By mutation of specific amino acids near the splice junctions, inteins can be converted into * Hai‑Feng Xia [email protected] 1
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
2
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
3
Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH 43210, USA
self-cleaving elements [8–10]. For instance, by mutation of the first residue of an intein (Cys) to Ala, a C-terminal cleavage reaction can be induced. Mutation of Asn to Ala at the end of the intein results in N-terminal cleavage. Inteins have been widely used in the field of biological technology and especially in the field of protein engineering, including in tagless protein purification, segmental isotopic labeling, biological sensors, and p
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