A point mutation in a murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli.
Recombinant DNA technology has made it possible to produce specific Fab and scFv antibody (Ab) fragments in prokaryotic host cells. Using vectors designed for periplasmic expression of encoded Ab fragments, we have been studying how the sequence and genetic localization of the light chain (L-chain) variable region gene of a mouse Ab (CB-Nm.1) determined the level of Ab production. The variable region was shown to belong to the V kappa V family and contained a previously unreported Ile72. Nine different Ab constructions were tested in monocistronic (scFv) or dicistronic (Fab) operons for their ability to affect the synthesis level of the L-chain. When the gene coding for the L-chain was located downstream from the Fd fragment gene, the substitution of codons encoding Ile by a codon encoding Thr was found to be crucial for any expression of the L-chain fragment. This was, however, not accompanied by an increase in L-chain-specific mRNA, neither was there any change in the size of the mRNA. The fact that the unmutated L-chain protein was produced from cells transformed with certain other constructions indicated that the protein as such was not incompatible with the prokaryotic environment. Together, this suggested that the translation process was involved in the restricted production of the L-chain. Thus, surprisingly small substitutions significantly affected the expression level, a fact that will have important implications on the library size expressed in prokaryotic hosts, including phage-displayed Ab libraries.
Dueñas
M
M
Division of Immunotechnology and Diagnostics, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Ayala
M
M
Vázquez
J
J
Ohlin
M
M
Söderlind
E
E
Borrebaeck
C A
CA
Gavilondo
J V
JV
D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007532:Isoleucine / Q000235:genetics; D008969:Molecular Sequence Data; D009876:Operon; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*
D004274:DNA, Recombinant; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007532:Isoleucine