Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties.
Escherichia coli (E. coli) is the most commonly used organism for expressing antibody fragments such as single chain antibody Fvs (scFvs). Previously, we have utilized E. coli to express well-folded scFvs for characterization and engineering purposes with the goal of using these engineered proteins as building blocks for generating IgG-like bispecific antibodies (BsAbs). In the study, described here, we observed a significant difference in the secondary structure of an scFv produced in E. coli and the same scFv expressed and secreted from chinese hamster ovary (CHO) cells as part of a BsAb. We devised a proteolytic procedure to separate the CHO-derived scFv from its antibody-fusion partner and compared its properties with those of the E. coli-derived scFv. In comparison to the CHO-derived scFv, the E. coli-derived scFv was found trapped in a misfolded, but monomeric state that was stable for months at 4 °C. The misfolded state bound antigen in a heterogeneous fashion that included non-specific binding, which made functional characterization challenging. This odd incidence of obtaining a misfolded scFv from bacteria suggests careful characterization of the folded properties of bacterially expressed scFvs is warranted if anomalous issues with antigen-binding or non-specificity occur during an engineering campaign. Additionally, our proteolytic methodology for obtaining significant levels of intact scFvs from highly expressed IgG-like antibody proteins serves as a robust method for producing scFvs in CHO without the use of designed cleavage motifs.
Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA.
Michelle C
MC
Vendel
Michael
M
Favis
William B
WB
Snyder
Flora
F
Huang
Allan D
AD
Capili
Jianying
J
Dong
Scott M
SM
Glaser
Brian R
BR
Miller
Stephen J
SJ
Demarest
D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics*; D016466:CHO Cells; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics*; D012995:Solubility
D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies