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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

Plasmid bearing the lysostaphin gene and over expression of this gene. A, plasmid construct for nisin-controlled lysostaphin production. PnisA, nisin-controlled promoter; matLss-2A, coding sequence for mature lysostaphin lacking the first 2 alanine residues; Term., transcription terminator; repC and repA, replication genes; lacF, food-grade lactose selection marker. B, SDS-PAGE showing the intracellular production of lysostaphin upon induction with nisin. 1, molecular weight marker; 2, cell extract without nisin-induction; 3, cell extract with induction with 10 ng/mL nisin; Lss, lysostaphin
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Figure 1: Plasmid bearing the lysostaphin gene and over expression of this gene. A, plasmid construct for nisin-controlled lysostaphin production. PnisA, nisin-controlled promoter; matLss-2A, coding sequence for mature lysostaphin lacking the first 2 alanine residues; Term., transcription terminator; repC and repA, replication genes; lacF, food-grade lactose selection marker. B, SDS-PAGE showing the intracellular production of lysostaphin upon induction with nisin. 1, molecular weight marker; 2, cell extract without nisin-induction; 3, cell extract with induction with 10 ng/mL nisin; Lss, lysostaphin

Mentions: The coding sequence of the lysostaphin gene of S. simulans biovar. Staphylolyticus, lacking its first two alanine residues [15], was cloned after PCR amplification into the NICE vector pNZ8148, resulting in plasmid pNZ1709 (see Table 1). The obtained construct was verified by nucleotide sequencing. Subsequently, the chloramphenicol-resistance cassette was exchanged for the lacF gene of L. lactis [11], leading to pNZ1710 (Fig. 1A). In this plasmid expression of the lysostaphin gene lss is under control of the nisin-inducible nisA promoter. Furthermore, this plasmid is selected by a food-grade mechanism, i.e. growth on lactose, and does not contain an antibiotic-resistance gene or its remnants. Lysostaphin, a 25-kD antibacterial protein, was produced in the cytoplasm of the cells. Figure 1B shows an SDS-PAGE image of the intracellular soluble protein fractions of NZ3900 (pNZ1710) before and after induction with nisin. After induction with nisin, lysostaphin is accumulated in the cell to about 10% of the soluble protein fraction, as estimated from SDS-PAGE (Figure 1B). Lysostaphin was isolated, purified (see below) and its N-terminal amino acid sequence was determined to be Thr-His-Glu-His-Ser-Ala [15]. This indicates that the correct protein was produced, that no significant intracellular degradation occurred and that the N-terminal formyl-methionine residue was removed.


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)

Plasmid bearing the lysostaphin gene and over expression of this gene. A, plasmid construct for nisin-controlled lysostaphin production. PnisA, nisin-controlled promoter; matLss-2A, coding sequence for mature lysostaphin lacking the first 2 alanine residues; Term., transcription terminator; repC and repA, replication genes; lacF, food-grade lactose selection marker. B, SDS-PAGE showing the intracellular production of lysostaphin upon induction with nisin. 1, molecular weight marker; 2, cell extract without nisin-induction; 3, cell extract with induction with 10 ng/mL nisin; Lss, lysostaphin
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Plasmid bearing the lysostaphin gene and over expression of this gene. A, plasmid construct for nisin-controlled lysostaphin production. PnisA, nisin-controlled promoter; matLss-2A, coding sequence for mature lysostaphin lacking the first 2 alanine residues; Term., transcription terminator; repC and repA, replication genes; lacF, food-grade lactose selection marker. B, SDS-PAGE showing the intracellular production of lysostaphin upon induction with nisin. 1, molecular weight marker; 2, cell extract without nisin-induction; 3, cell extract with induction with 10 ng/mL nisin; Lss, lysostaphin
Mentions: The coding sequence of the lysostaphin gene of S. simulans biovar. Staphylolyticus, lacking its first two alanine residues [15], was cloned after PCR amplification into the NICE vector pNZ8148, resulting in plasmid pNZ1709 (see Table 1). The obtained construct was verified by nucleotide sequencing. Subsequently, the chloramphenicol-resistance cassette was exchanged for the lacF gene of L. lactis [11], leading to pNZ1710 (Fig. 1A). In this plasmid expression of the lysostaphin gene lss is under control of the nisin-inducible nisA promoter. Furthermore, this plasmid is selected by a food-grade mechanism, i.e. growth on lactose, and does not contain an antibiotic-resistance gene or its remnants. Lysostaphin, a 25-kD antibacterial protein, was produced in the cytoplasm of the cells. Figure 1B shows an SDS-PAGE image of the intracellular soluble protein fractions of NZ3900 (pNZ1710) before and after induction with nisin. After induction with nisin, lysostaphin is accumulated in the cell to about 10% of the soluble protein fraction, as estimated from SDS-PAGE (Figure 1B). Lysostaphin was isolated, purified (see below) and its N-terminal amino acid sequence was determined to be Thr-His-Glu-His-Ser-Ala [15]. This indicates that the correct protein was produced, that no significant intracellular degradation occurred and that the N-terminal formyl-methionine residue was removed.

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