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Expanding the recombinant protein quality in Lactococcus lactis.

Cano-Garrido O, Rueda FL, Sànchez-García L, Ruiz-Ávila L, Bosser R, Villaverde A, García-Fruitós E - Microb. Cell Fact. (2014)

Bottom Line: In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism.Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species.These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. Olivia.Cano@uab.cat.

ABSTRACT

Background: Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement.

Results: We have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism.

Conclusions: Metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators.

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Related in: MedlinePlus

Yield of soluble (white bars) and IB rVP1GFP (grey bars) produced inL. lactisNZ9000 at 30°C.
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Fig3: Yield of soluble (white bars) and IB rVP1GFP (grey bars) produced inL. lactisNZ9000 at 30°C.

Mentions: The fluorescence emission (Figure 2) and the ratio of soluble versus insoluble protein (Figure 3) fractions were determined by fluorimetry and western blot (Figure 4), respectively. The fluorescence emission does not present significant differences at different production times. However, in general terms, it is possible to conclude that GFP activity increases at longer production times, being this effect more marked in the soluble fraction (Figure 2). Besides, it is worh mentioning that at 3 h post-induction, protein solubility reached 67%, a yield much higher than that reached in E. coli for the same protein under comparable conditions (10-18%, [34]). In this context, the potential of the system for the soluble protein production was confirmed with a difficult-to-express human catalase, which under these conditions majorly occurred at the soluble cell fraction (75%) (data not shown). On the other hand, the conformational quality, namely the ratio between protein activity and protein yield, of soluble rVP1GFP (estimated through its specific fluorescence) (Figure 5) evolved contrarily to the resulting protein amount in cells (Figure 2). This means that those conditions that favour protein solubility are those giving non-optimal results regarding conformational quality. Interestingly, such a divergence between protein yield and quality has been previously described in recombinant E. coli [29,35], but the present finding confirms this fact as a generic event.Figure 2


Expanding the recombinant protein quality in Lactococcus lactis.

Cano-Garrido O, Rueda FL, Sànchez-García L, Ruiz-Ávila L, Bosser R, Villaverde A, García-Fruitós E - Microb. Cell Fact. (2014)

Yield of soluble (white bars) and IB rVP1GFP (grey bars) produced inL. lactisNZ9000 at 30°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4308903&req=5

Fig3: Yield of soluble (white bars) and IB rVP1GFP (grey bars) produced inL. lactisNZ9000 at 30°C.
Mentions: The fluorescence emission (Figure 2) and the ratio of soluble versus insoluble protein (Figure 3) fractions were determined by fluorimetry and western blot (Figure 4), respectively. The fluorescence emission does not present significant differences at different production times. However, in general terms, it is possible to conclude that GFP activity increases at longer production times, being this effect more marked in the soluble fraction (Figure 2). Besides, it is worh mentioning that at 3 h post-induction, protein solubility reached 67%, a yield much higher than that reached in E. coli for the same protein under comparable conditions (10-18%, [34]). In this context, the potential of the system for the soluble protein production was confirmed with a difficult-to-express human catalase, which under these conditions majorly occurred at the soluble cell fraction (75%) (data not shown). On the other hand, the conformational quality, namely the ratio between protein activity and protein yield, of soluble rVP1GFP (estimated through its specific fluorescence) (Figure 5) evolved contrarily to the resulting protein amount in cells (Figure 2). This means that those conditions that favour protein solubility are those giving non-optimal results regarding conformational quality. Interestingly, such a divergence between protein yield and quality has been previously described in recombinant E. coli [29,35], but the present finding confirms this fact as a generic event.Figure 2

Bottom Line: In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism.Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species.These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. Olivia.Cano@uab.cat.

ABSTRACT

Background: Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement.

Results: We have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism.

Conclusions: Metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators.

Show MeSH
Related in: MedlinePlus