A new protocol for the isolation of key recombinant proteins in livestock production using lactic acid bacteria as a cell factory

• Published in: Journal of animal science 2016-10, Vol.94, p.77-77
• Main Authors: Gifre, L, Cano-Garrido, O, Fàbregas, F, Seras-Franzoso, J, Roca, R, Miralles, N Ferrer, Villaverde, A, Bach, A, Arís, A, Garcia-Fruitós, E
• Format: Article
• Language: English
• Subjects: Agglomeration; Animal sciences; Biological activity; Cells; Data processing; E coli; Epithelial cells; Escherichia coli; Gelatinase B; Interleukin 8; Interleukins; Lactic acid; Lactic acid bacteria; Lactococcus lactis; Lipopolysaccharides; Livestock; Livestock production; Mammary gland; Metalloproteinase; Probiotics; Proteins; Variance analysis
• ISSN: 0021-8812; 1525-3163
• DOI: 10.2527/jam2016-0159

Escherichia coli is one of the most widely used expression hosts for the production of recombinant proteins. However, obtaining pure and active proteins is not an easy task, especially considering difficult-to-express proteins, such as membrane or aggregation-prone proteins. Besides, E. coli contains lipopolysaccharide (LPS) that must be removed, which involves costly and time-consuming purification processes. Interestingly, Lactococcus lactis, which does not produce LPS, is able to form protein nanoclusters (aggregates) rich in the recombinant protein produced. The objective of this study was to develop an economically-affordable protocol to extract functional proteins from protein nanoclusters of L. lactis. For that, interleukin-8 (IL-8) stimulating protein (IL8SP), a difficult-to-express protein, and metalloproteinase 9 (MMP-9), an aggregation-prone protein, were used as model proteins. These proteins, that play important roles during the dry period of cows and have important economical potential, were recombinantly produced in L. lactis in the form nanoclusters. Next, IL8SP and MMP-9 nanoclusters were isolated and solubilized followed by some washing steps. Solubilized proteins were further purified following standard procedures for His-tagged proteins. Purified IL8SP and MMP-9 were quantified by Bradford assay and Western blot. The biological activity of IL8SP was measured in vitro by determining the expression of interleukin-8 (IL-8) in bovine mammary gland epithelial cell cultures. Specifically, cells were treated with 2 doses of IL8SP (9 and 90 μg). MMP-9 activity was determined by zymography. Data were analyzed using ANOVA. High aggregation ratios in nanoclusters were obtained for MMP-9 (99.24 ± 0.02%), whereas lower ratios were observed for IL8SP (37.32 ± 0.34%). Concerning biological activity, purified IL8SP showed a 1.6 and threefold-increase (P < 0.0001) of IL-8 expression, compared with the control cells, using 9 and 90 μg, respectively. Protein MMP-9 obtained with this protocol was also fully active when tested by zymography. In summary, these results show that it is possible to obtain soluble, pure and fully-active proteins from L. lactis protein-rich nanoclusters through a novel, cost-effective, and easy protocol.