Abstract
Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 β-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. β-lactamase was fused to the C-terminal of scFv and β-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for β-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.
Highlights
The Gram negative bacterium Escherichia coli is a mainstay of the biopharmaceutical industry, and is the most common non-mammalian cell production system for recombinant protein biopharmaceuticals[1]
Transformed E. coli BL21-A cultures were grown in terrific broth (TB) at 25 °C and recombinant protein production (RPP) was induced at an OD600 of 0.5 with 0.02% (w/v) arabinose to activate the pBAD promoter and 0.25% (w/v) glucose to regulate RPP by catabolite repression, conditions previously optimised for production of this scFv
Growth data revealed that induction of RPP inhibited growth of STII signal peptide (STIIsp)-scFv and DsbAsp-scFv cultures (Fig. 1a); CFU analysis corroborated this by way of decreased culturability, indicating stress (Fig. 1b), whereas PelBsp-scFv cultures displayed less growth inhibition and a lower decrease in culturability
Summary
The Gram negative bacterium Escherichia coli is a mainstay of the biopharmaceutical industry, and is the most common non-mammalian cell production system for recombinant protein biopharmaceuticals[1]. E. coli exploits multiple mechanisms for transport of proteins into the periplasm that include the SecB, SRP and twin-arginine (Tat) pathways (reviewed by[7,8]). Targeting of polypeptide chains to the periplasm via SecB, SRP or Tat requires an N-terminal signal peptide that interacts with components of the three pathways. This signal peptide is cleaved from the polypeptide chain by a protease during translocation, resulting in a mature protein in the periplasm. Multiple factors affect the functionality of the signal peptide It must interact, via electrostatic and hydrophobic interactions, with the inner membrane and the translocation apparatus to facilitate polypeptide transport[10]. The signal peptide affects protein translation and translocation via a variety of mechanisms
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