title: Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.
abstract: Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹⁴ cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve.
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Jiazhang
authors.initials: J
authors.lastname: Lian
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China; Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany.
authors.forename: Lei
authors.initials: L
authors.lastname: Kai
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: lhuangblue@zju.edu.cn.
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Peilin
authors.initials: P
authors.lastname: Cen
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: znxu@zju.edu.cn.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D020346:Aquaporins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002474:Cell-Free System; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000378:metabolism; D014158:Transcription, Genetic; D014867:Water / Q000378:metabolism
chemical_list: D020346:Aquaporins; D003902:Detergents; D029968:Escherichia coli Proteins; D008081:Liposomes; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C106425:aqpZ protein, E coli; D014867:Water; D012697:Serine Endopeptidases; C026515:type I signal peptidase
keywords: Aquaporin Z; Cell-free protein synthesis; Leader peptidase; Leader peptide; Membrane protein