Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.
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.
['Zhang|Xu|X|', 'Lian|Jiazhang|J|', 'Kai|Lei|L|', 'Huang|Lei|L|', 'Cen|Peilin|P|', 'Xu|Zhinan|Z|']
D000595:Amino Acid Sequence D020346:Aquaporins D002474:Cell-Free System D003902:Detergents D004926:Escherichia coli D029968:Escherichia coli Proteins D015971:Gene Expression Regulation, Enzymologic D005813:Genes, Synthetic D005822:Genetic Vectors D008081:Liposomes D008565:Membrane Proteins D008969:Molecular Sequence Data D014176:Protein Biosynthesis D021382:Protein Sorting Signals D011993:Recombinant Fusion Proteins D012697:Serine Endopeptidases D014158:Transcription, Genetic D014867:Water
Aquaporin Z Leader peptide Cell-free protein synthesis Membrane protein Leader peptidase