title: Expression of single-chain variable fragments fused with the Fc-region of rabbit IgG in Leishmania tarentolae.
abstract: BACKGROUND
  In 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).
  
  
  RESULTS
  Based 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.
  
  
  CONCLUSIONS
  To 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.
authors.forename: Mathias Lindh
authors.initials: ML
authors.lastname: Jørgensen
authors.forename: Niels Anton
authors.initials: NA
authors.lastname: Friis
authors.forename: Jesper
authors.initials: J
authors.lastname: Just
authors.forename: Peder
authors.initials: P
authors.lastname: Madsen
authors.forename: Steen Vang
authors.initials: SV
authors.lastname: Petersen
authors.affiliation: Department of Engineering, Aarhus University, Gustav Wieds Vej 10, Aarhus, Denmark. pk@mb.au.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Kristensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007797:Laminin / Q000276:immunology; D007891:Leishmania / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism*; D014746:Vimentin / Q000276:immunology
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007797:Laminin; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D014746:Vimentin