The food-grade bacterium L. lactis subsp. cremoris in conjunction with the Nisin Inducible Controlled Expression (NICE) system [1–3] has been extensively used over the last few decades as a valuable bacterial expression system for large-scale production of homologous or heterologous proteins , metabolic studies , or membrane proteins . The NICE system is based on the well characterized nisin-dependent, quorum-sensing mechanism of L. lactis [2, 3, 7]. It was initially exploited in L. lactis for heterologous protein overexpression and subsequently implemented in several other Gram-positive bacteria [2, 3, 7–10]. Typically, the genetically-engineered strain L. lactis subsp. cremoris NZ9000 is employed as expression host, as its chromosome contains the signal transduction genes nisR and nisK involved in the nisin-induced transcriptional control of the PnisA promoter . Any genes cloned downstream this nisin-inducible promoter PnisA can be expressed in a controlled manner upon addition of nisin to the bacterial culture . However, production of recombinant proteins can be problematic in L. lactis, as over-expressed proteins may be subject to poor expression, stability and/or solubility. Such drawbacks are intrinsically associated with the prokaryotic cell machinery limitations and therefore are inherent to all bacterial expression systems, representing a significant bottleneck in high level production of soluble proteins.
In E. coli, a 'microbial cell factory' of choice for producing heterologous proteins [11, 12], the development of the gene fusion technology proved to circumvent such recurrent and fundamental protein expression problems . This technology involves the linkage of the protein of interest with a carrier protein to generate a fusion protein. Addressing solutions to problematic protein expressions, many fusion expression systems have been engineered and successfully employed, using solubility-enhancing fusion partners such as Schistosoma japonicum glutathione-S-transferase (GST) , E. coli maltose binding proteins (MBP) , Staphylococcus protein A , E. coli N-utilization substance (NusA)  and E. coli thioredoxin (TrxA) [18, 19]. Along with the increasing number of fusion partners used, additional features have been successfully implemented to this technology, thus facilitating protein tagging, purification techniques and tag-mediated proteolytic cleavage [13, 20, 21]. The gene fusion technology provides a substantial palette of applications through the constant expansion of fusion gene expression systems available in E. coli. Nevertheless, the adaptation of these existing fusion partner systems to other expression hosts is sparse, even though significant progress has been made to develop new molecular tools and methods in alternative prokaryotic and eukaryotic expression systems [1, 22, 23]. The expression host L. lactis is currently lacking such a solubility-enhancing expression system to improve its spectrum of biotechnological applications, as L. lactis featured a number of benefits over other expression bacterial hosts, e.g. being a food-grade expression host, and the absence of endotoxins, extracellular proteinases and spores.
As part of our study on the structure-function analysis of lactococcal phage-host recognition and penetration, we attempted to over-express a number of proteins encoded by the lactococcal phage Tuc2009 in L. lactis. However, initial expression studies of individual protein subunits of Tuc2009 phage revealed such proteins often suffer from degradation, poor expression or result in insoluble protein aggregates, also called inclusion bodies (data not shown). The development of a fusion-based gene expression system in L. lactis could provide a novel strategy to express soluble proteins and avoid the use of laborious and spurious renaturation procedures. Among the numerous fusion partners employed, LaVallie et al. described the construction of an E. coli thioredoxin (TrxA) gene fusion system . In most cases, E. coli thioredoxin fusion proteins were soluble, correctly folded and biologically active . The E. coli thioredoxin thus appears to represent a good candidate for an L. lactis fusion-based gene expression system: small size of the fusion partner (11.67 kDa), ability to accumulate in a soluble form at high levels in the cytoplasm, steric accessibility of N- and C-termini of TrxA for protein fusions  and efficient generic protein purification methods available, i.e. immunoprecipitation or affinity chromatography [13, 24].
In the present study, we report on the construction of two new L. lactis thioredoxin-fusion gene expression vectors harbouring the nisin-controlled expression (NICE) system. We evaluated the efficiency of the newly-constructed fusion gene expression system, by producing individual proteins or protein complexes that initially could not be expressed or were not soluble in L. lactis. Our data indicate that the L. lactis thioredoxin-fusion vectors represent a very valuable addition to the L. lactis genetic toolbox, in particular for the over-production of soluble proteins.