摘要
侧孢短芽孢杆菌(Brevibacillus laterosporus)S62-9产生的抗菌肽(Brevibacillin,BBL)是一种非核糖体多肽,由非核糖体肽合成酶催化合成。本研究所用基因是从侧孢短芽孢杆菌(B.laterosporus)S62-9质粒基因组中克隆的一个非核糖体肽合成酶的腺苷化结构域(Nonribosomal peptide synthetase adenylation,NRPS-A)基因,根据此NRPS-A基因构建了pET21b-N1、pET21b-B2、pET22b-N1、pET22b-N3四个重组表达载体。将以上重组表达载体分别转化E.coli BL21、E.coli Rosetta(DE3)和E.coli Origami B(DE3)受体细胞,并用1 mmol/L异丙基β-d-硫代吡喃半乳糖吡喃糖苷(IPTG)在16°C 200 r/min下进行诱导,将诱导产物用SDS-PAGE检测分析。结果显示,4种重组载体在E.coliBL21中均无表达,在E.coliRosetta(DE3)大量表达但均以包涵体形式存在,在E.coli Origami B(DE3)均表达,但只有pET22b-N3这一重组表达载体出现了可溶性融合蛋白。可溶性融合蛋白对赖氨酸有较高的腺苷化活性,表明该A结构域负责抗菌肽BBL中的赖氨酸的识别和上载。该研究结果不仅为其它NRPS-A域的可溶性表达提供了方法,同时为研究抗菌肽BBL的生物合成机制提供依据。
The antibacterial peptide produced by Brevibacillus laterosporus S62-9 is a non-ribosomal polypeptide,which are synthesized by non-ribosomal peptide synthetase.In this study,a nonribosomal peptide synthetase adenylation(NRPS-A)gene was isolated from the putative gene cluster,and inserted into the prokaryotic expression vector pET21b-N1,pET21b-B2,pET22b-N1,and pET22b-N3.The above recombinant expression vectors were transformed into recipient cells of E.coli BL21,E.coli Rosetta(DE3),and E.coli Origami B(DE3),respectively.Then,the cells were induced with 1 mmol/L isopropylβ-d-thiogalactopyranoside(IPTG)and were further incubated at 16°C and 200 r/min,followed by SDS-PAGE detection and analysis.The results show that all of the four recombinant vectors were not expressed in E.coli BL21,but expressed in E.coli Rosetta(DE3)with a large quantities of inclusion bodies.Meanwhile,all of them were expressed in E.coli Origami B(DE3),but only the transformants containing the recombinant expression vector pET22b-N3 expressed the target protein in soluble form,and the size of soluble protein was consistent with the expectation.The soluble protein purified by Ni column showed the highest adenylation activity against lysine,Suggesting that the A domain is responsible for the recognition and uploading of lysine in BBL.The results not only provide a method for the soluble expression of other NRPS-A domains,but also provide a basis for studying the biosynthesis mechanism of Brevibacillus laterosporus S62-9 antibacterial peptide.
作者
刘欣
魏小雅
于宏伟
郭润芳
LIU Xin;WEI Xiaoya;YU Hongwei;GUO Runfang(College of Food Science and Technology,Hebei Agricultural University,Baoding 71000,China)
出处
《河北农业大学学报》
CAS
CSCD
北大核心
2020年第1期110-115,132,共7页
Journal of Hebei Agricultural University
基金
河北省自然科学基金(C2018204043).
