Department of Clinical Immunology, Institute of Pediatrics, Jagiellonian University Medical College, Wielicka str. 265, 30-663, Kraków, Poland.
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
Microb Cell Fact. 2018 Nov 16;17(1):177. doi: 10.1186/s12934-018-1028-2.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro.
The human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells.
We designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）选择性地消除肿瘤细胞。然而，这种分子的短生物学半衰期限制了其在临床上的潜在应用。我们的目的是构建一种无毒的乳酸乳球菌重组菌株作为载体，以有效和延长人类 TRAIL 的产生。在此，我们研究了导致体外产生生物活性蛋白的表达和分泌条件。
设计了具有优化密码子的人可溶性 TRAIL-cDNA（hsTRAIL-cDNA），以适应乳酸乳球菌宿主的密码子使用模式（密码子偏好性）。该 cDNA 构建体在乳球菌质粒分泌载体 pNZ8124 中合成并克隆，在 nisin 诱导的 PnisA 启动子的控制下表达。pNZ8124-hsTRAIL 质粒载体通过电穿孔转化到乳酸乳球菌 NZ9000 宿主菌株细胞中。在诱导过程中，当诱导剂浓度优化且存在丝氨酸蛋白酶抑制剂时，蛋白在中性 pH 下分泌。通过 Western blot 和氨基酸测序方法，我们发现 TRAIL 以两种形式分泌，表现为存在两个不同的分子大小带，都缺乏细菌信号肽 usp45 蛋白。通过 MTS 测定法，我们能够证明乳酸乳球菌上清液中存在的 hsTRAIL（hsTRAIL+）发酵液培养物对人 HCT116 结肠癌细胞具有细胞毒性，但对正常人类成纤维细胞没有毒性。流式细胞术分析显示 TRAIL 诱导了癌细胞凋亡。
我们设计了重组乳酸乳球菌，能够在体外高效产生生物活性的抗肿瘤人 TRAIL。在荷瘤 NOD-SCID 小鼠中的进一步研究将揭示 TRAIL 分泌的乳酸乳球菌是否可以作为该蛋白的安全载体，能够在体内诱导有效消除人结肠癌细胞。
