Intra- and extracellular expression of an scFv antibody fragment in E. coli: effect of bacterial strains and pathway engineering using GroES/L chaperonins.
We have studied the influence of bacterial host on the secretion of single-chain Fv antibody fragment (scFv), the production of this antibody fragment as intracellular fusion protein, and the effect of chaperonin coexpression on intracellular antibody expression. Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and the ompA leader sequence (ptrp/ompA/scFvCEA). Expression and secretion of this antibody fragment were highest in the W3110 strain, as determined by Western blot analysis and enzyme immunoassay, where the scFv fragment amounted to approximately 30% of the total periplasmic protein. Except for BMH71-18, the other strains were unsuitable for antibody fragment expression, suggesting screening of bacterial strains as an important parameter. For intracellular expression, the scFv was expressed as a fusion protein with a 26-amino acid N-terminal fragment of human interleukin-2 (IL-2), using the pIL-2f/scFvCEA vector. The fusion protein was expressed at 30% of total biomass and retained antigen binding after in vitro refolding. Co-expression of chaperonin-encoding plasmid pGroES/L with pIL-2f/scFv increased the intracellular production of the fusion protein twofold, with a similar increase in the final amount of active scFv antibody fragment that could be obtained after in vitro refolding. The chaperonins had no effect on secretion of scFv antibody fragments, using the ptrp/ompA/scFvCEA.
Dueñas
M
M
Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Vázquez
J
J
Ayala
M
M
Söderlind
E
E
Ohlin
M
M
Pérez
L
L
Borrebaeck
C A
CA
Gavilondo
J V
JV
D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000276:immunology; D018835:Chaperonin 10; D018834:Chaperonin 60; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D007124:Immunoenzyme Techniques; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013045:Species Specificity
D001426:Bacterial Proteins; D002272:Carcinoembryonic Antigen; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins