High-yield cell-free protein production from P-gel.
Cell-free systems represent a promising approach to quickly and easily produce preparative amounts of proteins. However, it is still challenging to obtain high volumetric yields (>mg ml(-1)) of proteins from the present cell-free systems. This protocol presents a cell-free protein synthesis method using a novel DNA gel that dramatically increases protein yield compared with current systems. This protein-producing gel (termed 'P-gel system' or 'P-gel'), which consists of genes as part of the gel scaffolding, can produce mg ml(-1) amounts of functional proteins. This protocol describes steps pertaining to plasmid design, fabrication of P-gel molds, formation of P-gel micropads and cell-free protein expression with an expected yield of up to 5 mg ml(-1) of functional Renilla luciferase (Rluc). This entire process can take 1-3 d, depending on the desired quantity of protein.
Park
Nokyoung
N
Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York, USA.
Kahn
Jason S
JS
Rice
Edward J
EJ
Hartman
Mark R
MR
Funabashi
Hisakage
H
Xu
Jianfeng
J
Um
Soong Ho
SH
Luo
Dan
D
D001671:Biochemistry / Q000379:methods; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry*; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005782:Gels / Q000737:chemistry*; D049410:Luciferases, Renilla / Q000096:biosynthesis* / Q000235:genetics; D014176:Protein Biosynthesis
D004129:Dimethylpolysiloxanes; D005782:Gels; C013830:baysilon; D004247:DNA; D049410:Luciferases, Renilla