| 1. |
- Lin, H. Y., et al.
(författare)
-
Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli
- 2004
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 87:5, s. 602-613
-
Tidskriftsartikel (refereegranskat)abstract
- Guanosine-3'5'-tetraphosphate (ppGpp) and as, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.
|
|
| 2. |
|
|
| 3. |
- Rozkov, Aleksei D., et al.
(författare)
-
Analysis and control of proteolysis of recombinant proteins in Escherichia coli.
- 2004
-
Ingår i: Advances in biochemical engineering/biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. ; 89, s. 163-195
-
Tidskriftsartikel (refereegranskat)abstract
- Proteolysis is one of the reasons for poor production of recombinant proteins in Escherichia coli. Important properties of E. coli proteases, which are relevant for the production of recombinant proteins, are reviewed. Furthermore, various strategies to control the proteolysis of the recombinant proteins are presented. These strategies for control of proteolysis can be applied on various stages of the process: design of more stable protein, a modification of the host cell in respect to proteolytic activity, optimisation of cultivation and downstream processing. However, before implementing these measures the proteolysis rate should be measured in order to calculate a potential benefit of reduced proteolysis rate.
|
|
| 4. |
- Syren, Per-Olof, et al.
(författare)
-
Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump
- 2007
-
Ingår i: Journal of chromatography. B. - : Elsevier B.V.. - 1570-0232 .- 1873-376X. ; 856, s. 68-74
-
Tidskriftsartikel (refereegranskat)abstract
- A parallel chromatog. procedure for the purifn. of milligram amts. of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quaternary ammonium group with respect to elution and regeneration properties and the 75 cm2 Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimized. By introducing this step into the protocol, endotoxin levels could be reduced approx. 100-fold to ≤5 EU/mg plasmid. The parallel procedure was set up on a multi-channel peristaltic pump and evaluated with four different vectors (2.7-11.5 kbp). Starting with 5-10 g of E. coli cell paste (wet wt.) generally satd. the membrane adsorber, resulting in plasmid DNA yields close to 10 mg. [on SciFinder(R)]
|
|
