Heterologous expression of Barnase and its mutants in Lactococcus lactis

Journal Information

J. Hefei Univ. Tech. (Nat. Sci.), Feb.2025, Vol.48 No.2: 245-250

DOI: 10.3969/j.issn.1003-5060.2025.02.016

Authors

AI Yaqian $ ^{1} $, MU Dongdong $ ^{1} $, LI Xingjiang $ ^{1} $, WANG Jin $ ^{2} $, WU Xuefeng $ ^{1} $

(1. School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China; 2. Anhui Xiqiang Dairy Group Co., Ltd., Huaibei 235099, China)

Abstract and Keywords

Abstract: In order to obtain a simple purified ribonuclease, the ribonuclease gene(Barnase) from Bacillus amyloliquefaciens BH072 was cloned into an inducible vector carrying the signal peptide Usp45 and then expressed in Lactococcus lactis. The molecular weight of Barnase was 14.6 kDa, which was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed the successful expression of the enzyme protein. To further improve the expression and activity of Barnase, the asparagine at position 58 of Barnase was mutated to histidine, and the expression time and inducer concentration were optimized. The optimal conditions were as follows: incubation time of 36 h and inducer concentration of 3 $ \mu $g/L, and the purified mutant enzyme activity was up to 3.7 kU/mL under these conditions.

Keywords: Barstar gene; polymerase chain reaction(PCR); protein expression; Lactococcus lactis

作者信息

艾雅倩 $ ^{1} $，穆冬冬 $ ^{1} $，李兴江 $ ^{1} $，王瑾 $ ^{2} $，吴学凤 $ ^{1} $

(1. 合肥工业大学食品与生物工程学院, 安徽合肥 230601; 2. 安徽曦强乳业集团有限公司, 安徽淮北 235099)

摘要和关键词

摘要: 为了得到纯化简便的核糖核酸酶,文章将来自解淀粉芽孢杆菌BH072的核糖核酸酶Barnase基因克隆到携带信号肽Usp45的诱导性载体中,然后将其导入乳酸乳球菌中进行表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)验证Barnase分子量大小为14.64 kDa,确认酶蛋白表达成功。为了进一步提高Barnase的表达量与活性,将Barnase的第58位天冬酰胺突变成组氨酸,并对表达时间和诱导剂质量浓度进行优化,得出最佳条件为孵育时间36 h、诱导剂质量浓度3 $ \mu $g/L,该条件下纯化突变体酶活性达到3.7 kU/mL。

关键词: Barstar 基因；聚合酶链式反应（PCR）；蛋白表达；乳酸乳球菌

Funding

安徽省科技重大专项资助项目（201903a06020007）；安徽省自然科学基金资助项目（2108085MC123）

合肥工业大学学报(自然科学版)

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