Deleting the first disulphide bond in an arenicin derivative enhances its expression in Pichia pastoris
Deleting the first disulphide bond in an arenicin derivative enhances its expression in Pichia pastoris
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DOI：
10.1111/lam.12770
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发表时间：
2017-09
影响因子：
2.4
通讯作者：
Na Yang;Xiumin Wang;D. Teng;R. Mao;Ya Hao;Xingjun Feng;Jianhua Wang
中科院分区：
文献类型：
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作者：
Na Yang;Xiumin Wang;D. Teng;R. Mao;Ya Hao;Xingjun Feng;Jianhua Wang
海洋抗菌肽NZ17074是Arenicola marina中Arenicin-3的变种，具有广泛的抗菌活性和高的生物利用度，可用于治疗细菌和真菌疾病。为了降低NZ17074的毒性，N6被设计成用丙氨酸取代第3和第20位的半胱氨酸，与小泛素样修饰物标签(SUMO)的C末端融合，并在酵母中表达。在发酵罐中诱导72h后，−1的产量可达921mgNZ1，是NZ17074的1.8倍。经30%甲酸裂解和Sephadex G-25柱层析纯化，每升发酵液中可获得9.7 mg重组多肽N6(RN6)，比NZ17074提高1.4−。与NZ17074相比，RN6对大肠杆菌、沙门氏菌、假单胞菌、葡萄球菌和链球菌的最小抑菌浓度分别为0·5、0·25-0·5、4、0·25-16和16μg ml−-1，显示出与NZ17074相似的抗菌活性。我们的结果表明，NZ17074中的第一个二硫键Cys3-Cys20不是抗菌活性所必需的，它的缺失可能会降低对宿主细胞的毒性。这些发现可能有助于设计新的抗菌肽，这些抗菌肽含有较少的二硫键，可能具有更强的活性。
The marine antimicrobial peptide NZ17074, a variant of arenicin‐3 from Arenicola marina that has broad antimicrobial activity and high bioavailability, can be designed to treat bacterial and fungal diseases. To reduce the toxicity of NZ17074, N6 was designed by replacing a cysteine in positions 3 and 20 with alanine, fused to the C‐terminus of the small ubiquitin‐like modifier tag (SUMO), and expressed in yeast. SUMO‐N6 yielded as much as 921 mg l−1 at 72 h after induction in a fermentor and increased 1·8–fold over SUMO‐NZ17074. After cleavage with 30% formic acid and purification by a Sephadex G‐25 column, 9·7 mg of the recombinant peptide N6 (rN6) was obtained from one‐litre fermentation broth, increasing 1·4−fold over NZ17074. Compared to NZ17074, rN6 displayed almost identical antimicrobial activity with a minimal inhibitory concentration of 0·5, 0·25–0·5, 4, 0·25–16 and 16 μg ml−1 against Escherichia, Salmonella, Pseudomonas, Staphylococcus and Streptococcus strains. Our results indicate that the first disulphide bond, Cys3‐Cys20, in NZ17074 is not necessary for antimicrobial activity and that its deletion might reduce toxicity to host cells. These findings may help design new antimicrobial peptides harbouring fewer disulphide bridges and may have more potent activity.