Expanding the recombinant protein quality in Lactococcus lactis

Abstract

BackgroundEscherichia 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.ResultsWe 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.ConclusionsMetabolic 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.

Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology

“…They described that under the overexpression of a fluorescent protein, highly fluorescent protein clusters are formed in Lactococcus lactis cytoplasm [18]. In this line, our group confirmed the presence of protein deposits in L. lactis cytoplasm using a modified green fluorescent protein [19]. The consideration of all these results together, lead us to believe that L. lactis might potentially be used for the generation of fully safe proteinbased particles that could be further explored as functional nanomaterials.…”

“…The electroporation mix was centrifuged for 10 min at 10,000Âg at 4°C and the pellet was resuspended in 100-200 lL of M17 media and plated. Besides, recombinant Green Fluorescent Protein (rGFP) previously described in [19,26] were also used.…”

A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays

“…These Gram-positive bacteria are non-pathogenic and non-invasive and inhabit different ecological niches (plant surfaces and the digestive tract of animals and human) 3 4, 5, 6, 7. Antigens delivered by recombinant LAB at the mucosal site would avoid the massive degradation of the antigens observed in the gut when purified antigens are used.…”

Lactococcus lactisharbouring Ara h 2.02 alleviates allergen‐specific Th2‐associated responses in sensitized mice

“…The efficiency of USP45 signal peptide was reported to vary dependent on the nature of the expressed protein and culture conditions (Dieye et al ; Hazebrouck et al ; Siaw et al ). Cano‐Garrido et al () reported the solubility of aggregation‐prone GFP variant (VP1GFP), expressed using pNZ8148 in L. lactis , was influenced by time, culture conditions and growth temperature, which have a dramatic effect not only on protein yield but also on protein solubility and conformational quality that were particularly favoured under fermentative anaerobic metabolism. Despite testing an extended duration of expression (6–12 h) at different growth temperatures (including the optimum 30°C), as previously reported for secretion of recombinant proteins containing USP45 (Subramaniam et al ; Siaw et al ), the expressed recombinant S‐Ara2.02‐H remained insoluble and intracellular even after 24 h of expression.…”

“…The efficiency of USP45 signal peptide was reported to vary dependent on the nature of the expressed protein and culture conditions (Dieye et al 2003;Hazebrouck et al 2007;Siaw et al 2016). Cano-Garrido et al (2014) reported the solubility of aggregation-prone GFP variant (VP1GFP), expressed using pNZ8148 in L. lactis, was Figure 3 Recombinant S-Ara2.02-H, rAra h 2.02 and nAra h 2 native peanut allergen showed comparable reactivity to serum IgE. A total of 5 µg ml À1 of the respective allergens were used to coat a 96-well microtitre plate for ELISA assay, using pooled sera from peanut allergic mice ( ) and biotinylated anti-mouse IgE for detection.…”