Le Loir Y, Gruss A, Ehrlich S D, Langella P
Laboratoire de Génétique Appliquée-URLEA, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy en Josas, France.
J Bacteriol. 1998 Apr;180(7):1895-903. doi: 10.1128/JB.180.7.1895-1903.1998.
Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only approximately 60% of total Nuc was present in a secreted form at approximately 5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to approximately 30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to approximately 80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to approximately 20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the alpha-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.
乳酸乳球菌是一种广泛应用于食品工业的革兰氏阳性菌，是分泌具有生物学用途蛋白质的潜在候选菌株。我们通过使用金黄色葡萄球菌核酸酶（Nuc）作为异源模型蛋白来检测乳酸乳球菌的分泌效率和能力。当从高效的乳球菌启动子及其天然信号肽在乳酸乳球菌中表达时，每升约5毫克的总Nuc中只有约60%以分泌形式存在。其余40%以细胞相关前体形式存在。通过删除Nuc天然前肽的17个残基（产生NucT），分泌效率进一步降低至约30%。我们鉴定出一种修饰方法，可提高天然Nuc和NucT的分泌效率。一种9个残基的合成前肽LEISSTCDA，在信号肽切割位点后立即融合，该前肽在+2和+8位置增加了两个负电荷。对于Nuc，通过插入LEISSTCDA，分泌效率提高到约80%，而不改变信号肽切割位点，产量提高了两到四倍（高达约每升20毫克）。NucT分泌效率的提高更为显著，从30%提高到90%。同样，第三种蛋白嗜热脂肪芽孢杆菌的α-淀粉酶的分泌效率也通过LEISSTCDA得到了提高。这些数据表明，LEISSTCDA合成前肽提高了乳酸乳球菌中不同异源蛋白的分泌。
