A new approach to obtain pure and active proteins from Lactococcus lactis protein aggregates

Abstract

The production of pure and soluble proteins is a complex, protein-dependent and time-consuming process, in particular for those prone-to-aggregate and/or difficult-to-purify. Although Escherichia coli is widely used for protein production, recombinant products must be co-purified through costly processes to remove lipopolysaccharide (LPS) and minimize adverse effects in the target organism. Interestingly, Lactococcus lactis, which does not contain LPS, could be a promising alternative for the production of relevant proteins. However, to date, there is no universal strategy to produce and purify any recombinant protein, being still a protein-specific process. In this context and considering that L. lactis is also able to form functional protein aggregates under overproduction conditions, we explored the use of these aggregates as an alternative source of soluble proteins. In this study, we developed a widely applicable and economically affordable protocol to extract functional proteins from these nanoclusters. For that, two model proteins were used: mammary serum amyloid A3 (M-SAA3) and metalloproteinase 9 (MMP-9), a difficult-to-purify and a prone-to-aggregate protein, respectively. The results show that it is possible to obtain highly pure, soluble, LPS-free and active recombinant proteins from L. lactis aggregates through a cost-effective and simple protocol with special relevance for difficult-to-purify or highly aggregated proteins.

The Biological Potential Hidden in Inclusion Bodies

“…Soluble MMP-9 was obtained from the solubilization of MMP-9 IBs produced in L. lactis, as previously described [12]. Briefly, bacteria were suspended (30:500 PBS:culture, volume) and disrupted for 4 rounds by French press, at 1500 psi, ice-coated, and with protease inhibitors (Complete EDTA-free, Roche), followed by a 2 h incubation in 0.05 mg/mL of lysozyme, at 37 • C, in agitation.…”

“…In most cases, IBs have been produced by using Escherichia coli as recombinant cell factory; however, their production has also been successfully done in lipopolysaccharide (LPS)-free recombinant systems, such as Lactococcus lactis [12,13].…”

Potential of MMP-9 based nanoparticles at optimizing the cow dry period: pulling apart the effects of MMP-9 and nanoparticles

“…Samples were tested for sterility by plating an aliquot in agar-M17 plates with 0.5% glucose and incubating them overnight at 30 °C. Aliquots of rMMP-9 IBs and mutant inactive rMMP-9 IBs were tested for purity through SDS-PAGE electrophoresis and Coomassie blue staining (75.7% purity obtained for MMP-9 IBs and 70.3% for mutMMP-9 IBs), and were quantified by interpolation with the bands obtained by a solubilized rMMP-9 as the standard 28 , using ImageJ software (version 1.46r, U. S. National Institutes of Health, Bethesda, Maryland, USA). The activity of the MMP-9 IBs and the inactivity of the mutMMP-9 IBs, were validated in vitro both by zymography and DQgelatin (data not shown).…”

“…Skimmed MGS were quantified for endogenous MMP-9 activity through zymography analysis. Solubilized MMP-9 28 was used to prepare an eight-point standard curve ranging from 400 to 25 ng. Sample supernatants were diluted 1/10 and diluted samples and standards were loaded with non-denaturing loading buffer into 10% SDS-PAGE gels containing 1% porcine gelatine.…”

Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles

“…The classic view of protein aggregates as mere inactive folding intermediates has been transformed into the idea of heterogeneous porous multimeric structures stabilized by a scaffold of cross beta-sheet structures that contain conformers of the recombinant protein in which a spectrum of recombinant protein species containing native-like conformations are incorporated [12]. In fact, it has been proven that biologically active protein species can be extracted from IBs by applying mild solubilization protocols, indicating the biologically active nature of proteins forming these aggregates [24]. In addition, further ground-breaking studies have suggested applications of IBs in nanomedicine.…”