Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli

Abstract

BackgroundDisulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations.ResultsHere we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active.ConclusionsOur results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0419-5) contains supplementary material, which is available to authorized users.

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM197

“…The plasmid pMJS205 containing the S. cerevisiae SOX Erv1p and H. sapiens PDI was obtained from Lloyd Ruddock, University of Oulu, Finland (Gaciarz et al 2016). It is based on pLysS and is pET-compatible.…”

Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories

“…From these selected gene fragments were amplified by PCR. The gene fragments were cloned with restriction digestion and ligation into a modified pET23-based vector with a pTac promoter replacing the T7 promoter [18] using the restriction site pairs NdeI/EcoRI for perlecan, NdeI/HindIII for mucin 2 and NdeI/BamHI for alpha tectorin. In all cases this resulted in an N-terminal hexahistidine tag prior to the first amino acid of the protein sequence (MHHHHHHM-).…”

“…Expression tests to screen for optimal conditions were carried out for the constructs in 24 deep well plates. The constructs were co-expressed together with CyDisCo components from pMJS205 [18] in up to four different E. coli strains (BL21(DE3) (from Stratagene), the Keio collection parental strain BW25113 [53], MDS42 [54] and BL21 (DE3) ∆trxA/trxC [55]), up to two different media (chemically defined [56] and terrific broth autoinduction media (Formedium) with trace elements and 0.8% glycerol and at two different temperatures (30 • C and 15 • C). All the constructs were found to be best expressed at 15 • C, in BL21(DE3) cells.…”

“…This system is based on heterologous co-or pre-expression of a eukaryotic catalyst of de novo disulfide bond formation (e.g., a sulfhydryl oxidase, most usually yeast Erv1p) as well as a eukaryotic catalyst of disulfide bond isomerization (e.g., a disulfide isomerase, most usually human PDI) i.e., catalysts of the two steps in native disulfide bond formation. Together these boost productive oxidative folding and hence the yield of disulfide-containing proteins [15][16][17][18].…”

“…The cytoplasm of E. coli has a reducing environment, preventing native disulfide formation, while the periplasm has a smaller volume [13] and may have limitations connected with the capacity of the translocation from the cytoplasm [14], which combined mean that periplasmic yields are usually significantly lower than cytoplasmic expression yields. To help to overcome the limitations of native disulfide bond formation in E. coli, we used synthetic biology to introduce a system known as CyDisCo (cytoplasmic disulfide bond formation in E. coli) [15][16][17][18]. This system is based on heterologous co-or pre-expression of a eukaryotic catalyst of de novo disulfide bond formation (e.g., a sulfhydryl oxidase, most usually yeast Erv1p) as well as a eukaryotic catalyst of disulfide bond isomerization (e.g., a disulfide isomerase, most usually human PDI) i.e., catalysts of the two steps in native disulfide bond formation.…”