Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against Hepatitis B.
A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene. Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv. Following production of the scFv protein using an E. coli expression vector (pGC), the scFv gene was recloned into a phage display vector (pHFA). This gene construct was introduced into E. coli mutator cells and the transformed cells were used as an inoculum for liquid cultures. After five cycles of growth at 37 degrees C, each followed by dilution and re-inoculation of fresh media, recombinant phage were recovered. Nucleotide sequence analysis of the scFv gene in phage selected on HBsAg-coated magnetic beads identified amino acid substitutions which produced an increase of greater than 10-fold in apparent production levels. Competitive ELISA studies showed that the selected scFv mutants appeared to have similar affinity to HBsAg as the parent scFv. The apparent increase in production was not the result of improved surface characteristics of regions uniquely exposed in scFvs, as the sites did not correlate with the variable/constant interface of the scFv variable region normally masked in Fabs or IgGs.
Coia
G
G
CSIRO Division of Biomolecular Engineering, CRC for Diagnostic Technologies, Parkville, Victoria, Australia. greg.coia@molsci.csiro.au
Ayres
A
A
Lilley
G G
GG
Hudson
P J
PJ
Irving
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006510:Hepatitis B Antibodies / Q000096:biosynthesis* / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006515:Hepatitis B virus / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics; D016296:Mutagenesis; D017422:Sequence Analysis, DNA
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv; D004247:DNA