Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies.
Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of considerable biotechnological and therapeutic potential. However, the reducing environment of the cytoplasm inhibits the formation of the intradomain disulfide bonds that are essential for correct folding and functionality of these antibody fragments. Thus, scFvs expressed in the cytoplasm are mostly insoluble and inactive.Here, we describe a general approach for stabilizing scFvs for efficient functional expression in the cell cytoplasm in a soluble, active form. The scFvs are expressed as C-terminal fusions with the Escherichia coli maltose-binding protein (MBP). We tested a large panel of scFvs that were derived from hybridomas and from murine and human scFv phage display and expression libraries by comparing their stability and functionality as un-fused versus MBP fused proteins. We found that MBP fused scFvs are expressed at high levels in the cytoplasm of E. coli as soluble and active proteins regardless of the redox state of the bacterial cytoplasm. In contrast, most un-fused scFvs can be produced (to much lower levels) in a functional form only when expressed in trxB(-) but not in trxB(+) E. coli cells. We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that they perform more efficiently in vivo as cytoplasmic intrabodies in E. coli. Thus, MBP seems to function as a molecular chaperone that promotes the solubility and stability of scFvs that are fused to it.
Bach
H
H
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Green Building, Room 202, Ramat Aviv 69978, Israel.
Mazor
Y
Y
Shaky
S
S
Shoham-Lev
A
A
Berdichevsky
Y
Y
Gutnick
D L
DL
Benhar
I
I
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D019793:Fluorescein; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D018832:Molecular Chaperones / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D058619:Maltose-Binding Proteins; D018832:Molecular Chaperones; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D001616:beta-Galactosidase; D019793:Fluorescein