Faculty of Life Science, KIM IL SUNG University, Pyongyang, Democratic People's Republic of Korea.
Faculty of Life Science, Kim Hyong Jik University of Education, Pyongyang, Democratic People's Republic of Korea.
Extremophiles. 2023 Aug 7;27(3):21. doi: 10.1007/s00792-023-01308-0.
Halocins, the proteinaceous antimicrobial agents produced by haloarchaea, may be used for the preservation of salted foods and the treatment of diseases. For their application and function explanation, it is necessary to produce the active recombinants. In this work, a haloarchaeal strain producing halocin was isolated from the salt-fermented shrimp and identified as Natrinema sp. RNS21 by 16S rRNA gene sequence analysis. From 1 L of RNS21 culture, about 0.32 mg of halocin with 96% purity was obtained. Based on the molecular weight, stability and amino acid sequence alignment, the antimicrobial peptide belonged to the halocin C8 (HalC8) family. HalC8 was expressed by fusion with glutathione-S-transferase (GST) in E. coli, followed by affinity purification and enterokinase (EK) cleavage. About 6.2 mg of recombinant HalC8 with 95% purity was obtained from 1 L of E. coli culture. MALDI-TOF-MS and RP-HPLC analysis indicated that the molecular weight and folding pattern of purified recombinant HalC8 were the same as those of native HalC8. Recombinant HalC8 showed obvious inhibitory activity against Haloferax volcanii. Contrast to native HalC8, the active recombinant HalC8 could be easily produced in a short time with a high yield.
卤虫素是由盐杆菌产生的蛋白类抗菌剂，可用于腌制食品的保存和疾病的治疗。为了应用和解释其功能，需要生产活性重组体。本工作从盐发酵虾中分离到一株产卤虫素的盐杆菌，通过 16S rRNA 基因序列分析鉴定为 Natrinema sp. RNS21。从 1 L RNS21 培养物中获得了约 0.32 mg、纯度为 96%的卤虫素。根据分子量、稳定性和氨基酸序列比对，该抗菌肽属于卤虫素 C8（HalC8）家族。通过与谷胱甘肽-S-转移酶（GST）融合在大肠杆菌中表达，然后进行亲和纯化和肠激酶（EK）切割。从 1 L 大肠杆菌培养物中获得了约 6.2 mg、纯度为 95%的重组 HalC8。MALDI-TOF-MS 和 RP-HPLC 分析表明，纯化的重组 HalC8 的分子量和折叠模式与天然 HalC8 相同。重组 HalC8 对 Haloferax volcanii 表现出明显的抑制活性。与天然 HalC8 相比，活性重组 HalC8 可以在短时间内以高产率轻易生产。
