Optimization of a mouse recombinant antibody fragment for efficient production from Escherichia coli.
A mutagenized mouse recombinant antibody fragment (rFab) that recognized HIV capsid protein was isolated from Escherichia coli at a level of 12 mg per liter of culture using standard shake flask methods. This is one of the highest yields of a modified antibody fragment obtained using non-fermentor-based methods. Recombinant Fab was isolated directly from the culture medium, which lacked complex materials such as tryptone and yeast extract. Fab isolated from the periplasm was not as homogeneous as that isolated directly from the culture medium. Optimization of the culture medium using recently developed media, the use of E. coli cell lines that contained rare tRNA codons, and mutagenesis of the Fab to improve the stability of the Fab were important factors in producing high-levels of the Fab. An isolation protocol easily adaptable to automation using a thiophilic-sepharose column followed by metal-chelate chromatography and the introduction of a non-traditional metal binding site for metal-chelate purification that bypasses the conventional hexahistidine tag cleavage step (to prevent the purification tag from interfering with crystallization) are additional features of this approach to produce a highly homogenous preparation of rFab. The resulting rFab binds to its antigen, p24, equivalent in character to the monoclonal from which the rFab was originally derived.
Nadkarni
Ashwini
A
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, Room 372, Wilson Pharmacy Building, University of Georgia, Athens, GA 30602-2352, USA.
Kelley
Laura-Lee Clancy
LL
Momany
Cory
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins