title: A cleavable self-assembling tag strategy for preparing proteins and peptides with an authentic N-terminus.
abstract: Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self-assembling peptides and a C-terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self-assembling peptide. Upon simple separation, the target protein or peptide with an authentic N-terminus is then released in the solution by intein-mediated cleavage. Different combinations of four self-assembling peptides (ELK16, L6KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI-CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI-CM, which has pH-shift inducible cleavage, was found to work well with three self-assembling peptides (L6KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx-strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N-terminus, which is especially effective for peptides of 30-100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, China.|Blue Moon Industrial Co. Ltd., Guangzhou, Guangdong, China.
authors.forename: Bihong
authors.initials: B
authors.lastname: Zhou
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, China.
authors.forename: Xianxing
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, China.|China National Petroleum & Chemical Planning Institute, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Xing
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, China.|School of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, China.
authors.forename: Zhanglin
authors.initials: Z
authors.lastname: Lin
mesh_terms: D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*
chemical_list: D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
keywords: Authentic N-terminus; C-terminal cleavage intein; Peptide preparation; Protein expression and purification; Self-assembling tag