High-yield production of diphtheria toxin mutants by high-density culture of C7 (beta)tox+ strains grown in a non-deferrated medium.
A high-density growth approach was utilized to produce mutated diphtheria toxin from two strains of Corynebacterium diphtheria: C7 (beta)(tox-201,tox-9) and C7 (beta)(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are CRM9 and CRM107; both of them carry the mutation in their binding site and, as a result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin. Since iron inhibits diphtheria toxin production, the traditional approach has been to grow the bacteria in a very low iron concentration. The procedure described here involved the use of a modified, non-deferrated, growth medium that provided fast and high-density growth of the bacteria, and which, when associated with simultaneous depletion of glucose and iron, enhanced the toxin production. Oxygen-enriched air was supplied to enable the bacteria to grow to a cell density giving an absorbance of 70 at 600 nm (15-20 g/l dry weight). The maximum toxin concentration in the culture supernatant was 150 mg/l. The CRM products, which remained stable following microfiltration and ultrafiltration, could be easily purified using a two-step chromatography procedure.
Fass
R
R
Biotechnology Unit, LCDB, NIDDK, NIH, Bethesda, MD 20892, USA.
Bahar
S
S
Kaufman
J
J
Shiloach
J
J
D003353:Corynebacterium diphtheriae / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media; D004167:Diphtheria Toxin / Q000096:biosynthesis*; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology; D009154:Mutation
D003470:Culture Media; D004167:Diphtheria Toxin; D007501:Iron; D005947:Glucose