Limits...
Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin.

Mierau I, Leij P, van Swam I, Blommestein B, Floris E, Mond J, Smid EJ - Microb. Cell Fact. (2005)

Bottom Line: Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production.The induction process was equally effective at all scales and yields of about 100 mg/L were obtained.Up-scaling was easy and required no specific effort.

View Article: PubMed Central - HTML - PubMed

Affiliation: NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands. igor.mierau@nizo.nl

ABSTRACT

Background: The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation.

Results: Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials.

Conclusion: This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.

No MeSH data available.


Related in: MedlinePlus

Purification of the overproduced lysostaphin. A, Typical chromatogram of a lysostaphin capture step. NaCl concentration (brown line) and absorption at 280 nm (blue line) are indicated. Fractions that were analysed by SDS-PAGE are indicated by grey bars underneath. The lysostaphin fraction (F6) is indicated with a black bar. B, SDS-PAGE analysis of the different fractions of the capture chromatography. 1, molecular weight marker; 2, cell extract before loading; 3, flow-through fraction (F3); 4, 5 and 6, fractions F4, F5 and F7; 7, lysostaphin fraction F6; 8, 9 and 10, fraction F6 diluted 1:2, 1:4 and 1:8.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1173137&req=5

Figure 4: Purification of the overproduced lysostaphin. A, Typical chromatogram of a lysostaphin capture step. NaCl concentration (brown line) and absorption at 280 nm (blue line) are indicated. Fractions that were analysed by SDS-PAGE are indicated by grey bars underneath. The lysostaphin fraction (F6) is indicated with a black bar. B, SDS-PAGE analysis of the different fractions of the capture chromatography. 1, molecular weight marker; 2, cell extract before loading; 3, flow-through fraction (F3); 4, 5 and 6, fractions F4, F5 and F7; 7, lysostaphin fraction F6; 8, 9 and 10, fraction F6 diluted 1:2, 1:4 and 1:8.

Mentions: A cation-exchange chromatography capture step was selected based on the relatively alkaline isoelectric point (pH 9.5) of lysostaphin [16]. Because of superior performance, the strong exchanger SP-Sepharose FF was chosen over the weak exchanger CM-Sepharose FF. Optimum lysostaphin binding was found at pH 7.5 in phosphate buffer. Lysostaphin was eluted using a NaCl step gradient at 0.5 M NaCl with the same pH and phosphate buffer concentrations (Methods) as used for loading (Figure 4A and 4B show an elution profile and SDS-PAGE analysis). Since maximum binding of lysostaphin was hindered by unknown components in the cell extract, the flow-through was re-fed to the column twice to capture more than 90% of lysostaphin from the cell extract. Finally, the eluate was diluted and all captured lysostaphin was applied to the column at once for an additional recapture step (Methods). The resulting material was ca. 90% pure lysostaphin as determined by SDS-PAGE analysis (Methods). The lysostaphin production yield in the fermentation was about 100 mg/l. Therefore about 300 g lysostaphin had been produced in each 3000-L fermentation run. The mean total yield of the downstream process was about 120 g, resulting in 40% recovery of the originally produced lysostaphin.


Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin.

Mierau I, Leij P, van Swam I, Blommestein B, Floris E, Mond J, Smid EJ - Microb. Cell Fact. (2005)

Purification of the overproduced lysostaphin. A, Typical chromatogram of a lysostaphin capture step. NaCl concentration (brown line) and absorption at 280 nm (blue line) are indicated. Fractions that were analysed by SDS-PAGE are indicated by grey bars underneath. The lysostaphin fraction (F6) is indicated with a black bar. B, SDS-PAGE analysis of the different fractions of the capture chromatography. 1, molecular weight marker; 2, cell extract before loading; 3, flow-through fraction (F3); 4, 5 and 6, fractions F4, F5 and F7; 7, lysostaphin fraction F6; 8, 9 and 10, fraction F6 diluted 1:2, 1:4 and 1:8.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1173137&req=5

Figure 4: Purification of the overproduced lysostaphin. A, Typical chromatogram of a lysostaphin capture step. NaCl concentration (brown line) and absorption at 280 nm (blue line) are indicated. Fractions that were analysed by SDS-PAGE are indicated by grey bars underneath. The lysostaphin fraction (F6) is indicated with a black bar. B, SDS-PAGE analysis of the different fractions of the capture chromatography. 1, molecular weight marker; 2, cell extract before loading; 3, flow-through fraction (F3); 4, 5 and 6, fractions F4, F5 and F7; 7, lysostaphin fraction F6; 8, 9 and 10, fraction F6 diluted 1:2, 1:4 and 1:8.
Mentions: A cation-exchange chromatography capture step was selected based on the relatively alkaline isoelectric point (pH 9.5) of lysostaphin [16]. Because of superior performance, the strong exchanger SP-Sepharose FF was chosen over the weak exchanger CM-Sepharose FF. Optimum lysostaphin binding was found at pH 7.5 in phosphate buffer. Lysostaphin was eluted using a NaCl step gradient at 0.5 M NaCl with the same pH and phosphate buffer concentrations (Methods) as used for loading (Figure 4A and 4B show an elution profile and SDS-PAGE analysis). Since maximum binding of lysostaphin was hindered by unknown components in the cell extract, the flow-through was re-fed to the column twice to capture more than 90% of lysostaphin from the cell extract. Finally, the eluate was diluted and all captured lysostaphin was applied to the column at once for an additional recapture step (Methods). The resulting material was ca. 90% pure lysostaphin as determined by SDS-PAGE analysis (Methods). The lysostaphin production yield in the fermentation was about 100 mg/l. Therefore about 300 g lysostaphin had been produced in each 3000-L fermentation run. The mean total yield of the downstream process was about 120 g, resulting in 40% recovery of the originally produced lysostaphin.

Bottom Line: Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production.The induction process was equally effective at all scales and yields of about 100 mg/L were obtained.Up-scaling was easy and required no specific effort.

View Article: PubMed Central - HTML - PubMed

Affiliation: NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands. igor.mierau@nizo.nl

ABSTRACT

Background: The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation.

Results: Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials.

Conclusion: This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.

No MeSH data available.


Related in: MedlinePlus