Differences in sequence-specific expression of two anti-arsonate Fabs in E. coli.
Monoclonal antibodies are potentially useful therapeutic agents and can now be produced in hosts such as bacteria. However, it has been found that bacterial expression of some antibody-combining site fragments is greatly diminished. We compared two homologous anti-arsonate antibodies, 36-65 and 36-71, to address the question of why the former but not the latter expresses well as Fab in E. coli. These antibodies are both derived from the same variable region germline genes but differ in affinity due to somatic mutations present in 36-71. To investigate the poor expression of 36-71 Fab, we examined several factors, such as cellular toxicity, induction with isopropylthio-beta-D-galactoside, and growth of transformed bacteria at lower temperatures (30 degrees C), as well as the possibility of E. coli strain-related expression of Fab. However, none of these factors made a significant difference to Fab expression. We next localized a significant portion of the defect in Fab expression to the heavy chain by swapping the heavy and light chains from the two antibodies to construct hybrid Fabs. We used site-directed mutagenesis to engineer amino acids into the variable regions of antibody 36-71, to reproduce those found in 36-65 which is expressed well in E. coli. The defect in expression is due to residues located in the complementarity-determining regions, as mutations of heavy chain framework residues to those present in 36-65 do not enhance expression of 36-71 Fab in E. coli.
Gill
D S
DS
Department Of Surgery, Massachusetts General Hospital, Charlestown, USA.
Wong
Y W
YW
Margolies
M N
MN
D000595:Amino Acid Sequence; D001152:Arsenicals / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D001152:Arsenicals; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; C054806:arsonic acid