Tuning recombinant protein expression to match secretion capacity

Abstract

Background: The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity.

Results: Here we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres.

Conclusions: The methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.

Keywords: Antibody fragments; Codon usage; Periplasmic secretion; Riboswitches; Signal peptides.

Fig. 1

Concept and workflow of applying the RiboTite expression system for titratable secretion. a The RiboTite system operates at both the transcription and translation level, to afford a gene regulatory cascade controlling both T7 RNAP and the gene of interest (GOI). Transcriptional control is mediated by the lacI repressor protein, induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Translational control is mediated by an orthogonal riboswitch (ORS) which releases and sequesters the ribsome binding site (RBS) in the presence and absence of the inducer Pyrimido-pyrimidine‐2,4‐diamine (PPDA) respectively. The system is composed on an E. coli expression strain BL21(LV2) strain, and expression plasmids containing the T7 promoter. Shown are the pENTRY and pDEST expression plasmids used that incorporate the signal peptide sequence (SP) to direct the produced protein for periplasmic translocation, and GOI and GOI-sfGFP fusions also under orthogonal riboswitch (ORS) and T7 promoter control. For further description of the BL21(LV2) cassette see Additional file 1: Fig. S1. b Riboswitch-dependent translation control of the RiboTite system is employed to match expression rate to the secretion capacity of the Sec pathway. c Schematic diagram of workflow. The pENTRY vectors were used to integrate the 5′UTR riboswitch with the 5′ encoded SP sequences. (1) A synonymous codon signal peptide library was generated, and (2–3) screened to select for clones that exhibit high protein expression and high regulatory control over basal induction. (4) Selected clones were sub-cloned into the pDEST vectors, and (5) screened for expression and secretion at small scale in shaker flasks (6) and in fed-batch bioreactors (7)

Fig. 2

scFvβ expresion control and correlation analysis for codon-optimised signal peptide sequences constructs. a Dose response curves for pENTRY constructs, relative fluorescent units normalized to cell density (RFU/OD₆₀₀) against inducer concentration for the WT and codon optimised DsbA and Piii signal peptide sequences. b Dose response curves for pDEST constructs, expression yield per cell reported as mg per g dry cell weight. c Linear regression of the pENTRY expression vs. pDEST expression for DsbA-E1 and Piii-E5. d Linear regression of the pENTRY expression vs pDEST secretion for the DsbA E1 and Piii E5. All data was taken from shaker flask expression under different inducer concentrations at 30 °C, 14 h post induction performed in biological triplicates

Fig. 3

Performance of different signal peptide-scFv pDEST from shake flask expression at 30 °C, 14 h post induction. Using the DsbA E1 signal peptide sequence (a, c, e) and the Piii E5 signal peptide sequence (b, d, f) expressing scFvβ (a–b), scFvT (c–d), and scFvH (e–f). The scFv yield plotted as mg per g dry cell weight (mg/g DCW) against inducer concentrations for different scFvs, quantified from western blot analysis, from the media (M), periplasm (PP) and spheroplast (SP) fractions. Performed in the absence of inducer (UI), or with the same IPTG (I) concentration (150 μM) and increasing PPDA concentrations (1.6–400 μM). All data was taken from shaker flask expression under different inducer concentrations at 30 °C, 14 h post induction performed in biological triplicates

Fig. 4

Western blot analysis of the periplasm (PP) and spheroplast (SP) samples from shaker flask induction. a–d Precursor protein processing and spheroplast retention of scFvβ and scFvT were assessed with either DsbA E1 or Piii E5 signal peptide. e–h The same periplasm fractions were also assessed for the disulfide bond formation under reducing and non-reducing conditions, with (+) or without (−) DTT

Fig. 5

Fed-batch fermentation cell growth, scFv protein yield and location. a, b Trial 1—scFvβ expressed with DsbA-E1 or Piii-E5 signal peptides samples collected at 4 h post-induction. c, d Trial 2—scFvβ or scFvT expressed with DsbA-E1 signal peptide samples collected at 4 h post-induction. The scFv yields plotted against inducer concretion (IPTG/PPDA) fixed IPTG (100 µM) and increasing PPDA concentrations (0, 4, 20, 40, 200, 400 µM) with scFv quantified from western blot analysis in the media (M), periplasm (PP) and spheroplast (SP) fractions. Protein production quantification was performed in technical triplicates

Fig. 6

SDS-PAGE and western blot analysis of Trial 2 fermentation samples. Coomassie stained SDS PAGE (upper pannel) and western blot analysis were performed on both spheroplast (SP) and periplasm (PP) samples of the pDEST-DsbA E1-scFvβ from 0 h and 4 h post-induction. The induction conditions were IPTG (100 µM) and increasing concentrations of PPDA (4, 40, 200 and 400 µM). The scFvβ protein was detected using the Anti-α His antibody. The E. coli anti-RNAP σ70 was used as a control and the signal was only detected in the SP cellular fractions demonstrating the correct cell fractionation procedure. Lysozyme (labelled) was used for the fractionation procedure