Expression of single-chain variable fragments fused with the Fc-region of rabbit IgG in Leishmania tarentolae

Abstract

BackgroundIn recent years the generation of antibodies by recombinant methods, such as phage display technology, has increased the speed by which antibodies can be obtained. However, in some cases when recombinant antibodies have to be validated, expression in E. coli can be problematic. This primarily occurs when codon usage or protein folding of specific antibody fragments is incompatible with the E. coli translation and folding machinery, for instance when recombinant antibody formats that include the Fc-region are needed. In such cases other expression systems can be used, including the protozoan parasite Leishmania tarentolae (L. tarentolae). This novel host for recombinant protein expression has recently shown promising properties for the expression of single-chain antibody fragments. We have utilised the L. tarentolae T7-TR system to achieve expression and secretion of two scFvs fused to the Fc-region of rabbit immunoglobulin G (IgG).ResultsBased on the commercial vector pLEXSY_IE-blecherry4 (Jena Bioscience; Cat. No. EGE-255), we generated a vector containing the Fragment Crystallisable (Fc) region of rabbit IgG allowing insertions of single chain antibody fragments (scFvs) in frame via Ncol/Notl cloning (pMJ_LEXSY-rFc). For the expression of rabbit Fc-fusion scFvs (scFv-rFc) we cloned two scFvs binding to human vimentin (LOB7 scFv) and murine laminin (A10 scFv) respectively, into the modified vector. The LOB7-rFc and A10-rFc fusions expressed at levels up to 2.95 mg/L in L. tarentolae T7-TR. Both scFv-rFcs were purified from the culture supernatants using protein A affinity chromatography. Additionally, we expressed three different scFvs without the rFc regions using a similar expression cassette, obtaining yields up to 1.00 mg/L.ConclusionsTo our knowledge, this is the first time that antibody fragments with intact Fc-region of immunoglobulin have been produced in L. tarentolae. Using the plasmid pMJ_LEXSY-rFc, L. tarentolae T7-TR can be applied as an efficient tool for expression of rFc fusion antibody fragments, allowing easy purification from the growth medium. This system provides an alternative in cases where antibody constructs express poorly in standard prokaryotic systems. Furthermore, in cases where bivalent Fc-fused antibody constructs are needed, using L. tarentolae for expression provides an efficient alternative to mammalian expression.

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

“…Here we show that even formats as complex as Camelidae IgG-like structures can over-expressing strains and optimized plasmids succeeded in yielding up to 4 mg/L of IgGs [15] and similar yields were obtained expressing scFv-Fc fusions in Leishmania tarantolae [31]. The protocol described in this article shows that it is possible to increase the yields to tens of mg/L and, according to recently published data [13], there is still the possibility to enhance the yields per liter of culture by increasing the cell density using optimized media and fermentation conditions.…”

A Novel Bispecific Antibody, S-Fab, Induces Potent Cancer Cell Killing

“…1,27-29 Among them, blinatumomab, a BiTE antibody that targets CD19 and CD3 by the fusion of 2 single-chain Fv antibodies, has already been approved for clinical use for B-cell leukemia. 11,30 However, single-chain Fv fusions, used by BiTE and other similar technologies, are generally less stable, 12 and difficult to express in bacteria, 14,15 and they have the tendency to aggregate. 13 Other formats, especially those based in IgG or Fab structures, are difficult to express in E. coli, making their production time consuming and unfeasible for less-equipped academic laboratories.…”

“…11 However, single-chain Fv fusions, used by BiTE and other similar technologies, have a few issues; they are generally less stable, 12 they have the tendency to aggregate, 13 and they are difficult to express in bacteria. 14,15 Many other formats, especially those based on IgG or Fab structures, are also difficult to express in bacteria, making protein engineering more challenging.…”

Upregulation of Mrps18a in breast cancer identified by selecting phage antibody libraries on breast tissue sections

“…In order to express soluble dAb-rFc, the sequence encoding BC5 antibody was sub-cloned from phagemid into pMJ_LEXSY-rFc vector, and expressed and purified as described [28]. …”