Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography.
The optimization of the production of recombinant DNA-derived proteins in Escherichia coli was investigated. We chose restriction endonucleases EcoRI and EcoRV from E. coli as model proteins, despite the observation that overproduction can result in a toxic effect to the cells. The enzymes were expressed as fusion proteins consisting of protein A from Staphylococcus aureus and the desired enzyme in order to facilitate purification. The expression of the fusion protein was induced by a temperature shift using the pR promoter of phage lambda regulated by the repressor plasmid pRK248cI. Data from batch fermentations provided the basis for planning a continuous two-stage fermentation. The EcoRI enzyme activity was investigated as a function of the induction time after cell disintegration and allowed an estimation of yield of the continuous culture. Plasmid instability, which was only observed under continuous conditions, could be prevented by adding tetracycline (resistance of the repressor plasmid) to the medium. We established a continuous cell disintegration system and purified the fusion protein semicontinuously by affinity chromatography. The biological activity of the fusion protein was the same as the native endonuclease so there was no need for cleavage of the fusion protein and the product could be used without further processing.
Beer
H D
HD
Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Federal Republic of Germany.
Maschke
H E
HE
Schügerl
K
K
D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D015246:Deoxyribonuclease EcoRI / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005285:Fermentation; D015870:Gene Expression; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000235:genetics
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases