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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

Comparison of growth characteristics of 1-L, 300-L and 3000-L cultures. Culture of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 at 1-L, 320-L and 3000-L scale with induction by 10 ng/mL nisin. As comparison, growth of an uninduced culture at 1-L scale is shown.
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Figure 3: Comparison of growth characteristics of 1-L, 300-L and 3000-L cultures. Culture of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 at 1-L, 320-L and 3000-L scale with induction by 10 ng/mL nisin. As comparison, growth of an uninduced culture at 1-L scale is shown.

Mentions: Lysostaphin production was scaled up from 1-L scale to 300-L scale and eventually 3000-L fermentations. Growth and induction conditions found at laboratory scale were directly transferred to the 300-L and 3000-L scale: Induction at an optical density OD600 = 1 with 10 ng/ml nisin. Details of media preparation for the larger scales are described in Methods. Figure 3 shows the growth characteristics of induced cultures of L. lactis carrying the plasmid pNZ1710 at all three scales. All three cultures behaved very similarly, despite the difference in scale of more than 3 orders of magnitude. The lysostaphin yields of the 1-L and 3000-L fermentations after induction were approximately 100 mg/L. Four consecutive 3000-L production runs were carried out to produce raw material for further down stream processing. The growth characteristics of the four fermentation runs were virtually identical. In each run approximately 300 g lysostaphin (100 mg/L) were produced and subsequently used as starting material for purification.


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)

Comparison of growth characteristics of 1-L, 300-L and 3000-L cultures. Culture of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 at 1-L, 320-L and 3000-L scale with induction by 10 ng/mL nisin. As comparison, growth of an uninduced culture at 1-L scale is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Comparison of growth characteristics of 1-L, 300-L and 3000-L cultures. Culture of L. lactis NZ3900 containing the lysostaphin expression plasmid pNZ1710 at 1-L, 320-L and 3000-L scale with induction by 10 ng/mL nisin. As comparison, growth of an uninduced culture at 1-L scale is shown.
Mentions: Lysostaphin production was scaled up from 1-L scale to 300-L scale and eventually 3000-L fermentations. Growth and induction conditions found at laboratory scale were directly transferred to the 300-L and 3000-L scale: Induction at an optical density OD600 = 1 with 10 ng/ml nisin. Details of media preparation for the larger scales are described in Methods. Figure 3 shows the growth characteristics of induced cultures of L. lactis carrying the plasmid pNZ1710 at all three scales. All three cultures behaved very similarly, despite the difference in scale of more than 3 orders of magnitude. The lysostaphin yields of the 1-L and 3000-L fermentations after induction were approximately 100 mg/L. Four consecutive 3000-L production runs were carried out to produce raw material for further down stream processing. The growth characteristics of the four fermentation runs were virtually identical. In each run approximately 300 g lysostaphin (100 mg/L) were produced and subsequently used as starting material for purification.

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