Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control

Bäcklund, Emma; Markland, Katrin; Larsson, Gen

doi:10.1007/s00449-007-0144-x

Cited by 18 publications

(18 citation statements)

References 24 publications

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“…The negative effects of acetic acid on growth and recombinant protein production have been shown before [19] and accumulation needs to be avoided. Different strains of E. coli are known to produce different amounts of acetic acid even though their growth rate is similar.…”

Section: Discussionmentioning

confidence: 99%

Optimisation of surface expression using the AIDA autotransporter

2011

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BackgroundBacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.ResultsThe use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface-anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l-1 could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.ConclusionsA number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

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Section: Discussionmentioning

confidence: 99%

Optimisation of surface expression using the AIDA autotransporter

2011

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“…The resulting expression vectors, all expressing the proteins from the arabinose promoter, were transformed into E. coli BL21(DE3) (derived from the original Novagen stock), AF1000 (MC4100 relA + ), PPA668 ( ptsM ), PPA652 ( ptsG ) and PPA689 ( ptsG, ptsM ) [8]. …”

Section: Methodsmentioning

confidence: 99%

“…The medium was supplemented with 1 ml/l trace element solution [8] and1 ml/l 1 M MgSO 4 and 10 g/l glucose. 100 μg/ml carbenicilin was added to cells containing plasmids.…”

Section: Methodsmentioning

confidence: 99%

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Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli

2011

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BackgroundThe production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes.ResultsA set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities.ConclusionsThe use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

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“…Much of the effort aimed at increasing recombinant protein production in bacterial strains have been directed at maximizing the biomass production with high cell density cultivation method [6][7][8][9][10] and little is known about the effects of media composition on the expression of recombinant proteins. The composition of the growth medium at or during the induction phase can significantly affect heterologous protein expression.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation

Babaeipour

Abbas

Sahebnazar

et al. 2009

Bioprocess Biosyst Eng

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Development of inexpensive and simple culture media is always favorable for recombinant protein over-expression in E. coli. The effects of medium composition on the production of recombinant human granulocyte-colony stimulating factor (rh-GCSF) were investigated in batch culture of E. coli BL21 (DE3) [pET23a-hgcsf]. First, the optimum medium for production of rh-GCSF was determined; and, then it was shown that mixture of amino acid addition at induction time, which was determined on the basis of amino acids frequency in the recombinant protein, increases recombinant protein expression level significantly. Furthermore, the effect of glucose concentration on productivity of rh-GCSF was investigated; 20 g/l of glucose will result in maximum attainable biomass and rh-GCSF in this process. At optimum conditions, a cell dry weight of 10.5 g/l, an expression level of about 35% of total cellular protein, rh-GCSF concentration of 1.75 +/- 0.1 g/l, and overall rh-GCSF yield of 165 +/- 5 mg/g were obtained.

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Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control

Cited by 18 publications

References 24 publications

Optimisation of surface expression using the AIDA autotransporter

Optimisation of surface expression using the AIDA autotransporter

Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli

Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation

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