Efficient bacterial production of functional antibody fragments using a phagemid vector.
The so-called 'in vitro evolutionary method' using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 degrees C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3-15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.
Kuba
Hiroyoshi
H
Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Furukawa
Atsuko
A
Okajima
Toshihide
T
Furukawa
Koji
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics*; D002151:Calorimetry; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D019151:Peptide Library; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins