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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

Growth characteristics of 1-L culture. Culture at 1-L scale of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 with and without induction by 10 ng/mL nisin.
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Figure 2: Growth characteristics of 1-L culture. Culture at 1-L scale of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 with and without induction by 10 ng/mL nisin.

Mentions: Induction of lysostaphin production was carried out at an optical density at 600 nm of about 1 (light path 1 cm) (mid exponential growth phase) (0.3 g/L cell dry weight [21]) by adding nisin (Figure 2). After induction, lysostaphin production proceeded for 6 – 8 hours. Figure 2 shows that upon induction, growth of the culture is severely inhibited. This is likely the result of lysostaphin accumulation in the cell that appears to have growth inhibiting properties (viable plate counts drop within 20 min after induction 3–4 orders in magnitude).


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)

Growth characteristics of 1-L culture. Culture at 1-L scale of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 with and without induction by 10 ng/mL nisin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Growth characteristics of 1-L culture. Culture at 1-L scale of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 with and without induction by 10 ng/mL nisin.
Mentions: Induction of lysostaphin production was carried out at an optical density at 600 nm of about 1 (light path 1 cm) (mid exponential growth phase) (0.3 g/L cell dry weight [21]) by adding nisin (Figure 2). After induction, lysostaphin production proceeded for 6 – 8 hours. Figure 2 shows that upon induction, growth of the culture is severely inhibited. This is likely the result of lysostaphin accumulation in the cell that appears to have growth inhibiting properties (viable plate counts drop within 20 min after induction 3–4 orders in magnitude).

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