Subtilisin QK-2: secretory expression in Lactococcus lactis and surface display onto gram-positive enhancer matrix (GEM) particles

Abstract

BackgroundPurified from the supernatant of Bacillus subtilis QK02 culture broth, Subtilisin QK-2 is a type of effective thrombolytic reagent that has great exploitable potential. However, the unbearable flavor that occurs with fermentation and the complicated methods that are required to obtain pure products limit the application of this enzyme. Lactic acid bacteria (LAB)-based delivery vehicles are promising as cheap and safe options for medicinal compounds. The secretory expression and surface display using LAB may popularize Subtilisin QK-2 more easily and conveniently with minimal adverse effects.ResultsSubtilisin QK-2 was expressed successfully in two forms using lactic acid bacteria. For the secretory expression in Lactococcus lactis, Subtilisin QK-2 was efficiently secreted into the culture using the promoter PnisA and signal peptide SPUsp. The expression levels were not different in L. lactis NZ9000 and NZ3900 without the effect of different selection markers. However, leaky expression was only detected in L. lactis NZ3900. The biological activity of this secreted Subtilisin QK-2 was enhanced by modulating the pH of medium to slightly alkaline during induction and by codon optimization of either the entire gene sequence (qk′) or only the propeptide gene sequence (qkpro′). For surface display onto gram-positive enhancer matrix (GEM) particles, n LysM repeats from the C-terminal region of the major autolysin AcmA of L. lactis were fused to either the C-terminus (n = 1, 3, 5) or the N-terminus (n = 1) of the Subtilisin QK-2. These fusion proteins were secreted into the culture medium, and the QK-3LysM was able to bind to the surface of various LAB GEM particles without a loss of fibrinolytic activity. Furthermore, the binding capacity significantly increased with a higher concentration of QK-3LysM. Compared to the free-form Subtilisin QK-2, the QK-3LysM displayed on the surface of GEM particles was more stable in the simulated gastric juice.ConclusionsCombined with the safety and popularity of LAB, Subtilisin QK-2 may be easily applied worldwide to prevent and control thrombosis diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0478-7) contains supplementary material, which is available to authorized users.

A review on Lactococcus lactis: from food to factory

“…Antigens fused to streptococcal protein anchor enable them to be docked onto the peptidoglycan of GEM particles, which was also shown to elicit an immune response in nasally immunized mice [35]. A similar approach was also employed recently for subtilisin QK-2 using GEM [36]. The drawback of this system, however, is that the lactococcal cells are merely a carrier of the displayed protein, not a factory producing the proteins, thus repeated introduction of proteins displayed on lactococcal cells may be needed.…”

“…This suggested a possible role for L. lactis as a cell factory for colorectal cancer therapeutics [88]. Other examples of therapeutics produced using L. lactis as a microbial cell factory include subtilisin QK-2 as an anti-thrombotic agent [36], BT crystal protein Cry5B as an anthelminthic [89], heat shock protein (hsp) 65-6IA2P2 against type 1 diabetes [90] and many others as summarized in Table 2.…”

Surface-displayed porcine reproductive and respiratory syndrome virus from cell culture onto gram-positive enhancer matrix particles

“…First, researchers revealed that subtilisin QK-2 (QK) was successfully separated using GEM surface display technology. In detail, QK-LysM, as a fusion protein, was loaded onto GEM particles by the LysM domain [18]. In other studies, a similar strategy was used to successfully attach various viral antigens, including porcine circovirus type two cap protein [15] and porcine epidemic diarrhea virus N protein [7], to GEM particles.…”

Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Lactococcus lactis Cell Factories

“…The plasmids used in these studies have pSH71-based replicons with the Usp45 signal peptide sequence for protein secretion fused with the gene of interest, and the addition of three LysM repeats in the surface-displayed version. The surface displayed proteins in L. lactis were considered more stable and with higher bioactivity than the secreted counterparts [ 57 , 58 ]. In a different study from Ma et al [ 59 ], the authors administered to chicken live L. lactis expressing cytoplasmic, secreted or surface anchored Eimeria tenella 3-1E protein.…”