Enhancing the secretory yields of leech carboxypeptidase inhibitor in Escherichia coli: influence of trigger factor and signal recognition particle.
The signal recognition particle (SRP) dependent secretion pathway is as an attractive alternative to Sec-dependent export for the production of disulfide-bonded and/or fast-folding recombinant proteins in the Escherichia coli periplasm. SRP, which shares a ribosomal attachment site with the molecular chaperone trigger factor (TF), recognizes highly hydrophobic signal sequence as they emerge from the ribosome and delivers ribosome nascent chain complexes to FtsY for subsequent cotranslational translocation of target proteins across the SecYEG pore. However, like in the case of Sec-dependent export, secretory yields can be limited by the accumulation of precursor proteins in the cytoplasm. Using leech carboxypeptidase inhibitor (LCI) fused to the SRP-dependent DsbA signal sequence as a model system, we show that a null mutation in the gene encoding TF (Deltatig) or SRP co-expression reduce pre-LCI accumulation by half, and that quantitative export can be achieved by combining the two strategies. Interestingly, enhanced precursor processing did not alter periplasmic LCI levels but increased the amount of protein excreted in the growth medium. All mature LCI was nearly fully active and an 80% increase in productivity was achieved in Deltatig cells alone due to their faster growth. Our results show that competition between SRP and TF can interfere with efficient export of recombinant proteins targeted to the SRP pathway and establish TF-deficient strains and SRP co-expression as a simple solution to improve yields.
Puertas
Juan-Miguel
JM
Department of Chemical Engineering, University of Washington, Seattle, WA 98195-1750, USA.
Nannenga
Brent L
BL
Dornfeld
Kevin T
KT
Betton
Jean-Michel
JM
Baneyx
François
F
D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D047709:Hirudo medicinalis / Q000235:genetics*; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018271:Signal Recognition Particle / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; C116332:LCI protein, Hirudo medicinalis; D011506:Proteins; D018271:Signal Recognition Particle; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli