Enhanced secretion of recombinant proteins via signal recognition particle (SRP)-dependent secretion pathway by deletion of rrsE in Escherichia coli.
Although signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion, its limited capacity for the protein secretion is a major hurdle for utilizing the pathway as an attractive route for secretory production of recombinant proteins. Therefore, we developed an Escherichia coli mutant, whose efficiency of secretion via the SRP pathway was dramatically increased. First, we developed a novel FACS-based screening system by combining a periplasmic display system (PECS) and direct fluorescent labeling with the organoarsenic compound, FlAsH-EDT2 . With this screening system, transposon-insertion library of E. coli was screened, and then we isolated mutants which exhibited higher protein production through the SRP pathway than the parental strain. From the genetic analysis, we found that all isolated mutants had the same mutation-disruption of the 16S rRNA gene (rrsE). The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP-dependent proteins, antibodies, and G protein-coupled receptor. For the large-scale production of IgG and GPCR, we performed fed-batch cultivation with the rrsE-deficient mutant, and very high yields of IgG (0.4 g/L) and GPCR (1.4 g/L) were obtained. Biotechnol. Bioeng. 2016;113: 2453-2461. © 2016 Wiley Periodicals, Inc.
Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Yong Jae
YJ
Lee
Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Roojin
R
Lee
Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Se Hwa
SH
Lee
Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Sung Sun
SS
Yim
Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. kjjeong@kaist.ac.kr.|KAIST Institute for the BioCentury, Yuseong-gu, Daejeon, Republic of Korea. kjjeong@kaist.ac.kr.
Ki Jun
KJ
Jeong
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D055785:Gene Knockdown Techniques / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods*; D009154:Mutation / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D055571:Secretory Pathway / Q000235:genetics*; D018271:Signal Recognition Particle / Q000235:genetics*
D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D018271:Signal Recognition Particle
16S ribosomal RNA; Escherichia coli; FACS; G protein-coupled receptor (GPCR); SRP-dependent secretion