Efficient expression of aquaporin Z in Escherichia coli cell-free system using different fusion vectors.
Aquaporin Z (AqpZ) is a typical orthodox aquaporin with 6 transmembrane domains and five connecting loops. In order to express this complex membrane protein efficiently, E. coli cell-free expression system was employed as an alternative to produce aquaporin Z. Using different fusion vectors containing AqpZ gene, the expression level of fusion proteins in cell-free system varied from 7.97 to 578.35 microg/ml, while 7.34 to 340.81 microg/ml for target protein (AqpZ). The free energy of mRNA secondary structure at translation initiation region (TIR) was predicted and demonstrated a positive relationship with the expression level of AqpZ in cell-free system. This is the first report of expressing water channel protein in E. coli cell-free system, which has become a highly promising tool for fast and efficient production of integral membrane proteins.
Xu
Zhinan
Z
Institute of Bioengineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China.
Lian
Jiazhang
J
Cai
Jin
J
D020346:Aquaporins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D002474:Cell-Free System; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012270:Ribosomes; D013698:Templates, Genetic; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site
D020346:Aquaporins; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C517609:TrxA protein, E coli; C106425:aqpZ protein, E coli; D006639:Histidine; D013879:Thioredoxins; D008274:Magnesium