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

L Gifre-Renom,R Roca-Pinilla,À Bach,F Fàbregas,E Garcia-Fruitós,O Cano-Garrido,J Seras-Franzoso,A Villaverde,M Devant,N Ferrer-Miralles,A Arís

doi:10.1038/s41598-018-32213-8

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.

Highlights

Recombinant proteins represent a growing market and their applications are numerous for human medicine[1] and animal health and production[2]
In a first approach to evaluate the production profile of mammary serum amyloid A3 (M-SAA3) in L. lactis, the production kinetics of this model protein was analyzed at 30 °C at different times post-induction
The separation of the soluble and the insoluble fractions of the cell lysate indicated that M-SAA3 was mainly produced in the soluble form (65–80%) in L. lactis cytoplasm, protein aggregates (IBs) were formed (Fig. 1a)

Summary

Introduction

Recombinant proteins represent a growing market and their applications are numerous for human medicine[1] and animal health and production[2]. L. lactis aggregates (or inclusion bodies -IBs-), as it occurs with those found in other recombinant expression systems, are fully functional protein nanoclusters that are spontaneously formed under overproduction conditions[9,18,19] The formation of such protein deposits is relevant for those proteins difficult-to-purify and/or prone-to-aggregate[9,18]. The recombinant protein is free of LPS, the strategy used to produce and purify each specific protein is still largely protein-specific, as it occurs in other expression systems Since these aggregates might be an alternative source of difficult-to-obtain proteins in L. lactis and considering the need to develop a universal protocol for the production and purification of LPS-free recombinant proteins, the objective of this study was to develop a broad-application strategy to extract functional protein from L. lactis aggregates. MMP-9 is an enzyme that degrades the extracellular matrix and is involved in the immune response and tissue remodeling

Objectives

Methods

Results

Discussion

Conclusion