title: Subtilisin QK-2: secretory expression in Lactococcus lactis and surface display onto gram-positive enhancer matrix (GEM) particles.
abstract: BACKGROUND
  Purified 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.
  
  
  RESULTS
  Subtilisin 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 P nisA 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.
  
  
  CONCLUSIONS
  Combined with the safety and popularity of LAB, Subtilisin QK-2 may be easily applied worldwide to prevent and control thrombosis diseases.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China.
authors.forename: Ruifeng
authors.initials: R
authors.lastname: Mao
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China.
authors.forename: Kangping
authors.initials: K
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China.
authors.forename: Zhenwei
authors.initials: Z
authors.lastname: Han
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China. wangyefu@whu.edu.cn.
authors.forename: Yefu
authors.initials: Y
authors.lastname: Wang
mesh_terms: D015153:Blotting, Western; D003062:Codon; D005342:Fibrinolysis; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003062:Codon; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D020860:Subtilisin
keywords: Gram-positive enhancer matrix; Lactic acid bacteria; Secretory expression; Subtilisin QK-2; Surface display; Thrombus