Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coli.
Historically, the general secretory (Sec) pathway of Gram-negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin-arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N-termini upon reaching the periplasm and (iii) proteins fused to maltose-binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well-folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
Adam C
AC
Fisher
Jae-Young
JY
Kim
Ritsdeliz
R
Perez-Rodriguez
Danielle
D
Tullman-Ercek
Wallace R
WR
Fish
Lee A
LA
Henderson
Matthew P
MP
DeLisa
D058947:Bacterial Secretion Systems; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D058947:Bacterial Secretion Systems; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli
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