为高效制备可溶性人源Nanog蛋白,降低Nanog蛋白的使用成本。该研究通过分子克隆技术构建重组大肠杆菌(pET32a-Nanog),利用IPTG诱导进行可溶性融合表达。采用镍离子柱亲和层析法纯化产物并将纯化后产物进行肠激酶酶切后得到目的蛋白,Western blot鉴定该蛋白抗体结合的特异性并进一步优化发酵条件。结果表明,原核表达载体pET32a-Nanog构建成功,可在大肠杆菌中与硫氧还蛋白融合表达,融合蛋白经肠激酶酶切后得到相对分子质量约为38 kDa的蛋白,Western blot鉴定该蛋白可与Nanog抗体特异性结合。在摇瓶培养条件下,得到最优发酵条件是诱导温度37 ℃、IPTG浓度0.2 mmol/L、甘油为补料碳源;15 L发酵罐上进一步优化溶氧条件及温度控制方式,最终在溶氧设置在30%条件下,37 ℃(诱导期间30 ℃维持30 min)发酵12 h后得到最高Nanog产量1 386.4 mg/L,经蛋白纯化后纯度达到90%,为后续Nanog多克隆抗体的制备和基础医学实验研究奠定了基础。
To efficiently prepare soluble human-derived Nanog protein and reduce the cost, recombinant expression Escherichia coli(pET32a-Nanog) was constructed by molecular cloning technology and soluble fusion expression was induced by isopropyl-β-D-thiogalactopyranoside(IPTG). The product was purified by Ni-NTA affinity chromatography and was digested by enterokinase to obtain the target protein. Western blot was used to identify the specificity of antibody binding to the protein and the fermentation conditions were further optimized. The results showed that the prokaryotic expression vector pET32a-Nanog was successfully constructed and could be fused expression with the thioredoxin in E.coli. The fusion protein was digested by enterokinase to obtain a protein with a relative molecular mass of about 38 kDa. The protein could bind to Nanog antibodies specifically by Western blot. The optimal fermentation results under shake culture conditions contained induction temperature of 37 ℃, IPTG concentration 0.2 mmol/L, glycerin as the feed carbon source. The dissolved oxygen and temperature control methods were further optimized in a 15 L fermenter. The highest Nanog yield was up to 1 386.4 mg/L after 12 hours with the dissolved oxygen set at 30%, 37 ℃(30 ℃ for 30 min during the induction period), and reached 90% purity after protein purification. This study provided the base for the subsequent preparation of Nanog polyclonal antibodies and Nanog basic medical experimental research.
[1] BOOTH H A,HOLLAND P W.Eleven daughters of Nanog[J].Genomics,2004,84(2):229-238.
[2] EZEH U I,TUREK P J,REIJO R A,et al.Human embryonic stem cell genes Oct4,Nanog, Stellar, and GDF3 are expressed in both seminoma and breast carcinoma[J].Cancer,2005,104(10):2 255-2 265.
[3] HYSLOP L,STOJKOVIC M,ARMSTRONG L,et al.Downregulation of Nanog induces differentiation of human embryonic stem cells to extraembryonic lineages[J].Stem Cells (Miamisburg),2005,23(8):1 035-1 043.
[4] LIU Na,LU Min.The signal transduction pathways and molecules for ES cells self-renewal[J].科学通报(英文版),2005,50(8):721-726.
[5] SILVA J,NICHOLS J,THEUNISSEN T W,et al.Nanog is the gateway to the pluripotent ground state[J].Cell,2009,138(4):722-737.
[6] PRADO M M,FRAMPTON A E,STEBBING J,et al.Gene of the month:Nanog [J].Journal of Clinical Pathology,2015,68(10):763-770.
[7] PAN Qiong,MENG Linkun,YE Jun,et al.Transcriptional repression of miR-200 family members by Nanog in colon cancer cells induces epithelial-mesenchymal transition (EMT)[J].Cancer Letters,2017,392(1):26-38.
[8] HYSLOP L,STOJKOVIC M, ARMSTRONG L, et al.Downregulation of Nanog induces differentiation of human embryonic stem cells to extraembryonic lineages[J].Stem Cell,2010,23(8):1 035-1 043.
[9] YOU Linping,GUO Xin,HUANG Yuenan.Correlation of cancer stem-cell markers Oct4,Sox2,and Nanog with clinicopathological features and prognosis in operative patients with rectal cancer[J].Yonsei Medical Journal,2018,59(1):35-40.
[10] SHAHINI A,MISTRIOTIS P,ASMANI M,et al.Nanog restores contractility of mesenchymal stem cell-based senescent microtissues[J].Tissue Engineering Part A,2017,23(11-12):535-545.
[11] 李军,吕长荣,窦琳,等.小鼠Nanog基因原核表达载体的构建及表达[J].中国农业科学,2007,40(2):373-378.
[12] 张经余,王爱娥,李美香,等.人NanogP8蛋白的原核表达及多克隆抗体的制备[J].中国生物工程杂志,2009,29(3):30-35.
[13] SU Linqian,JIANG Qi,YU Lingang,et al.Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C[J].Microbial Cell Factories,2017,16(1):24-30.
[14] BRUNA A,VICTÓRIA M,FELIPE O,et al.Production of recombinant β-galactosidase in bioreactors by fed-batch culture using DO-stat and linear control[J].Biocatalysis and Biotransformation,2019,37(1):3-9.
[15] RUAN ALESSANDRO, REN Chang,QUAN Shu.Conversion of the molecular chaperone Spy into a novel fusion tag to enhance recombinant protein expression[J].Journal of Biotechnology,2020,307(none):131-138.
[16] ZHANG Hucheng,YANG Jun,YANG Guowei,et al.Production of recombinant protein G through high-density fermentation of engineered bacteria as well as purification[J].Molecular medicine reports,2015,12(2):31-32.
[17] PUGH R J.Bubble and foam chemistry [M].Cambridge:Cambridge University Press,2016:331-371.
[18] RAÙL E P,JOSÉ G S,DIAZ E N,et al.Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis[J].Electronic Journal of Biotechnology,2018,33(2):16-23.
[19] MAN Zaiwei,RAO Zhiming,CHENG Yipeng,et al.Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed[J].World Journal of Microbiology and Biotechnology,2014,30(2):661-7.
[20] YANG Yalin,TIAN Zigang,DA Teng,et al.High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression[J].Journal of Biotechnology,2009,139(4):326-331.
[21] 王传振,刘晓鹏,崔怡,等.猪转录因子Nanog高效表达、多克隆抗体制备及其应用[J].农业生物技术学报,2016,24(1):35-43.
[22] 苏鹏,龚国利.优化大肠杆菌表达外源蛋白的研究进展[J].生物技术通报,2017,33(2):16-23.
[23] 高霖,朱莉,杨泽林,等.高分子质量聚唾液酸生产菌株诱变筛选及其发酵优化[J].食品与发酵工业,2019,45(10):22-28.
[24] CHEN X,LIU L,LI J,et al.Improved glucosamine and N-acetylglucosamine production by an engineered Escherichia coli via step-wise regulation of dissolved oxygen level[J].Bioresource Technology,2012,110(none):534-538.
