水蛭素变体Ⅲ (Hirudin varidant Ⅲ,Hv3)是一种从水蛭中提取的活性成分,在预防和治疗白内障方面具有潜在作用。为了制备足量Hv3用于进一步的临床前应用研究,该研究开发了一种在谷氨酸棒杆菌(Corynebacterium glutamicum)中高效、稳定表达重组水蛭素变体Ⅲ(recombinant Hirudin varidant Ⅲ, Rhv3)的办法。首先,在C.glutamicum中构建含Ptac启动子和CspA信号肽的Rhv3分泌表达菌株,比较其在不同培养基中的表达情况。结果表明在BHI培养基中Rhv3活性最高,达到3.02×103 ATU/L。为进一步提升Rhv3产量,通过核糖体结合位点(ribosome binding site,RBS)筛选,选用RBS1应用于Rhv3多拷贝菌株构建和表达。然后利用3A组装技术构建含不同信号肽和不同拷贝数的Rhv3分泌表达菌株进一步优化其产量。通过放大发酵培养,Rhv3产量达到1.89 g/L,活性达到10.91×103 ATU/L。最后利用镍柱对Rhv3进行简单纯化,其纯度达到90%。该异源表达策略有效提高Rhv3表达量,为Rhv3的重组生产提供了一种质量可靠、低成本的表达体系。
Hirudin variant Ⅲ (Rhv3), a leech derived active ingredient, has the potential effect on the prevention and treatment of cataracts. To prepare sufficient amounts of Rhv3 for clinical research, the effective and stable process for Rhv3 expression in Corynebacterium glutamicum was developed in this study. Firstly, Rhv3 secretion expression strain, which was controlled by Ptac promoter and CspA signal peptide, was constructed in C. glutamicum. The expression level of this strain was compared in different media. The results showed that Rhv3 activity was highest in the BHI medium and reached 3.02×103 ATU/L. Next, the RBSs was screened using egfp reporter gene to increase production of recombinant protein. And the RBS1 was finally selected and applied to the construction and expression of the strain of multi-copy strain of Rhv3. By screening the signal peptide and copy number using 3A assembly, the six was selected as the best copy number of Rhv3. It was found that this strain could promote the expression level to the maximum extent in C. glutamicum. Further increased production of Rhv3 was achieved in large-scale fermentation, the M6S-PorB strain was used to produce Rhv3. Its yield was approximately accumulate 1.89 g/L, and the activity of Rhv3 reached 10.91×103 ATU/L, which was 3.61 times higher than that of the starting strain. Finally, the Rhv3 was purified by nickel column, and the purity was higher than 90%. To the best of our knowledge, this is the first report of multi-copy strategy to produce recombinant protein in C. glutamicum. This strategy based on the 3A assembly would provide an expression system with effective, reliable, and low cost for recombinant protein expression in C. glutamicum.
[1] ZHANG J G, LAN N N.Hirudin variants production by genetic engineered microbial factory[J].Biotechnology & Genetic Engineering Reviews, 2018, 34(2):261-280.
[2] 赖宝英, 陈志峰.水蛭素对急性脑出血患者血脑屏障紧密连接蛋白表达和患者颅内压的影响[J].黑龙江中医药, 2021, 50(5):107-108.
LAI B Y, CHEN Z F.Effects of hirudin on expression of blood brain barrier tight junction protein and intracranial pressure in patients with acute cerebral hemorrhage[J].Heilongjiang Journal of Traditional Chinese Medicine, 2021, 50(5):107-108.
[3] LI T, MA J Y, XU Z H, et al.Transcriptomic analysis of the influence of methanol assimilation on the gene expression in the recombinant Pichia pastoris producing hirudin variant 3[J].Genes, 2019, 10(8):606.
[4] HUANG C C, ZHANG X R, QU J, et al.Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization[J].Journal of Industrial Microbiology and Biotechnology, 2012, 39:1487-1494.
[5] GIDDINGS G, ALLISON G, BROOKS D, et al.Transgenic plants as factories for biopharmaceuticals[J].Nature Biotechnology, 2000, 18:1151-1155.
[6] KIM M D, HAN K C, KANG H A, et al.Coexpression of BiP increased antithrombotic hirudin production in recombinant Saccharomyces cerevisiae[J].Journal of Biotechnology, 2003, 101(1):81-87.
[7] KIM M D, LEE T H, LIM H K, et al.Production of antithrombotic hirudin in GAL1-disrupted Saccharomyces cerevisiae[J].Applied Microbiology and Biotechnology, 2004, 65(3):259-262.
[8] LYU J, HUANG C C, ZHANG X R, et al.Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310 mut2 signal peptide[J].Biotechnology Letters, 2012, 34(1):61-65.
[9] RADZIO R, KÜCK U.Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum[J].Applied Microbiology and Biotechnology, 1997, 48(1):58-65.
[10] ROSENFELD S A, NADEAU D, TIRADO J, et al.Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris[J].Protein Expression and Purification, 1996, 8(4):476-482.
[11] MENDOZA-VEGA O, HEBERT C, BROWN S W.Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain:Physiological considerations during the bioprocess design[J].Journal of Biotechnology, 1994, 32(3):249-259.
[12] MATSUI T, SATO H, YAMAMURO H, et al.High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production[J].Journal of Biotechnology, 2008, 134(1-2):88-92.
[13] MATSUI T, SATO H, YAMAMURO H, et al.High cell density cultivation of recombinant E.coli for hirudin variant 1 production by temperature shift controlled by pUC18-based replicative origin[J].Applied Microbiology and Biotechnology, 2008, 80:779-783.
[14] KAWAGUCHI H, HASUNUMA T, OHNISHI Y, et al.Enhanced production of γ-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation[J].Microbial Cell Factories, 2021, 20:228.
[15] ZHANG Y C, LIU Y D, ZHANG S Y, et al.Metabolic engineering of Corynebacterium glutamicum WM001 to improve l-isoleucine production[J].Biotechnology and Applied Biochemistry, 2021, 68(3):568-584.
[16] WANG Y, GAO X, LIU X, et al.Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants Ⅲ in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2020, 104:8257-8266.
[17] VAN DER REST M E, LANGE C, MOLENAAR D.A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA[J].Applied Microbiology and Biotechnology, 1999, 52:541-545.
[18] 梁伟锋, 张朝春, 杨希才.一种多拷贝毕赤酵母表达载体的构建及人脑源性神经营养因子的表达[J].微生物学报, 2005, 45(1):34-38.
LIANG W F, ZHANG C C, YANG X C.Construction of a multi-copy Pichia expression vector and expression of human brain-derived neurotrophic factor in Pichia pastoris[J].Acta Microbiologica Sinica, 2005, 45(1):34-38.
[19] SEYED HOSSEINI FIN N A, BARSHAN-TASHNIZI M, SAJJADI S M, et al.The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E.coli[J].Protein Expression and Purification, 2019, 157:42-49.
[20] LIN Y J, HUANG L H, HUANG C T.Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence[J].PLoS One, 2013, 8(3):e59099.
[21] ZHONG Z X, XU Z N, PENG L, et al.Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli[J].Applied Microbiology and Biotechnology, 2006, 71(5):661-667.
[22] 周伟杰, 吴凤梅, 姚冬生, 等.通过毕赤酵母表达系统获得高纯度的重组人血管内皮生长因子(rhVEGF165)[J].生物工程学报, 2021, 37(11):4083-4094.
ZHOU W J, WU F M, YAO D S, et al.Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris[J].Chinese Journal of Biotechnology, 2021, 37(11):4083-4094.
