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1 生物工程学报 Chin J Biotech 28, March 25; 24(3): Chinese Journal of Biotechnology ISSN Institute of Microbiology, CAS & CSM, All rights reserved 研究报告 BMP6 雷荣悦, 乔玉欢, 闫继东, 杨爽, 朱天慧, 371 : BMP6 是一种调节成骨细胞和成软骨细胞分化的骨诱导因子, 在修复各种骨缺损方面具有很好的应用潜力 有诱骨活性的 BMP6 是多二硫键的二聚体蛋白, 疏水性极强容易聚集沉淀 为了在大肠杆菌中可溶表达具有生物活性的重组人 BMP6(rhBMP6), 构建了具有 TRX GST MBP CBD 融合标签和 His 6 标签的 rhbmp6 成熟肽原核表达载体, 调节诱导温度和 IPTG 浓度, 比较不同融合标签和诱导条件对目的蛋白表达量和溶解性的影响 结果表明, MBP 最能有效的增强 rhbmp6 的溶解性, 诱导条件对溶解性影响较小 大肠杆菌 BL21 trxb(de3) 这种硫氧还蛋白还原酶缺陷菌株为 rhbmp6 二硫键在胞质中形成提供了合适的氧化还原环境 MBP 和 BL21 trxb(de3) 相结合在细胞质中高效可溶表达出了 BMP6 融合蛋白二聚体 表达产物经亲和层析和凝胶排阻层析纯化后, 能诱导成肌细胞系 C2C12 向成骨细胞方向转化 : BMP6, MBP, BL21 trxb(de3), 可溶性 Soluble Expression of Recombinant Human BMP6 in Escherichia coli and Its Purification and Bioassay in Vitro Rongyue Lei, Yuhuan Qiao, Jidong Yan, Shuang Yang, and Tianhui Zhu Laboratory of Medical Molecular Genetics, College of Medicine, Nankai University, Tianjin 371, China Abstract: BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His 6 -tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active. Keywords: BMP6, MBP, BL21 trxb(de3), solubility BMPs(bone morphogenetic proteins) Urist,, (BMPs) [1] 2, 21 Received: June 26, 27; Accepted: August 16, 27 Supported by: the Tianjin Natural Science Foundation (No. 5YFJMJC18). Corresponding author: Tianhui Zhu. Tel: ; 天津市自然科学基金资助 (No. 5YFJMJC18)

2 : BMP6 453 BMPs BMPs,, ( ) ( ) [2,3] BMPs N C, 3, BMP6 BMPs, 6, cdna 2943bp, 1539 bp, , 15.6 kd, pi 8.6, BMP6,, BMPs, BMP6 [4,5] BMP6, BMPs,,, ;, ;,,, [6] BMPs, IPTG,, rhbmp6,, 1 材料与方法 1.1 质粒, 菌株和细胞株 pgex-bmp6, pet-15b-bmp6, pet-32a, pmal-c2x,pmal-p2x, ptwin1 New England Biolabs DH5α XL1-blue BL21(DE3) BL21trxB(DE3), TB1 ER2566 New England Biolabs, LB C2C12, 1%FBS DMEM, 5 % CO 2, 工具酶和试剂 T4DNA, Xa New England Biolabs, DNA OMEGA, ECL Western Blot Pharmacia, Anti-BMP6 Santa Cruz Biotechnology, Anti-goat IgG-HRP Promega, BSA DOC DTT Glycine Urea TritonX-1 Sigma, Millipore, IPTG TaKaRa( ), Superdex-75 1/3GL GE Healthcare Inc, Amylose Resin New England Biolabs 1.3 表达载体的构建 mrna, oligo(dt) 18, PCR BMP6 DNA (1299~ 1718 nt, GenBank accession No. NM1718), DNA, pgex-bmp6, pet-15b-bmp6, pgex-bmp6, pet-15b-bmp6, BMP6 cdna, pet-32a, pmal-c2x, pmal-p2x, ptwin1, 1.4 诱导表达和可溶性比较 E. coli, 37, 3 d 1:1 2 ml LB, OD 6.5, IPTG,, : =1:1 (5 mmol/l Tris-Cl; ph 7.4; 2 mmol/l NaCI;.5 mmol/l EDTA), 2%, 2, 4 s, 3 s,, 4 C 12 r/min 3 min SDS-PAGE,,

3 454 ISSN1-361 CN /Q Chin J Biotech March 25, 28 Vol.24 No.3 Table 1 Recombinant plasmid 表 1 大肠杆菌表达载体的构建 Schematic representation of constructs cloned into various vectors for expression in E. coli Restriction endonuclease Fusion protein Expression host Fusion size (kd) Characteristic pgex-5x-bmp6 EcoR I/Xho I GST DH5a 26 Glutathione, efficient translation initiation pmal-c2x-bmp6 EcoR I/Xba I MBP TB1 42 High-level expression, a one-step purification using MBP s affinity for maltose pmal-p2x-bmp6 EcoR I/Xba I MBP TB1 42 The signal peptide on pre-mbp directs fusion proteins to the periplasm allowing folding and disulfide bond formation pet-15b-bmp6 Nco I/Xho I 6 * His BL21(DE3) 2.4 Ni-NTA purification, small and easy to add pet-32a-bmp6 Nco I/Xho I TRX BL21(DE3) 2 Efficient translation initiation, enhance solubility ptwin1-bmp6 Nco I/Xho I CBD-Intein ER Chitin bead resin purification, ph- and temperaturedependent cleavage of the intein 1.5 SDS-PAGE 及 Western blot 鉴定 Tris-Tricine / SDS-PAGE (12 V, 16 min), Univ Unvi SDS-PAGE, 5% TBST anti-bmp6 anti-goat IgG 2 h, ECL 1.6 融合蛋白 MBP-BMP6 的纯化及 Factor Xa 酶切鉴定 BMP6 MBP, Amylose,.45 μm, 4 Amylose, 1, 1 mmol/l, Superdex-75 1/3GL, :.2 ml/min 1.5 ml,, 28 nm, 1 ml, SDS-PAGE MBP-BMP6: Factor Xa, Factor Xa BMP6 Factor Xa MBP- BMP6.5:1, 4 C 48 h 1.7 碱性磷酸酶的活性测定, C2C12 BMPs,,, C2C12 DMEM, 37, 5% CO 2 8%~9%, 5 d, MTT, PBS 3, 5 μl, 2 min, 45 nm OD,, 6, Microsoft Excel 2 结果 2.1 表达载体在不同诱导条件下表达及可溶性检测, ;, :,, GST 3% SDS-PAGE, GST-BMP6,, [7] BL21(DE3)/ pet-15b-bmp6 IPTG, SDS-PAGE, 1%,, BL21(DE3)/ pet-32a-bmp6,

4 : BMP %, ( 1) BMP6, pmal-c2x- BMP6 TB1, SDS-PAGE, 57 kd, MBP-BMP6 3%, MBP-BMP6 ( 4) 图 1 SDS-PAGE 分析.1 mmol/l IPTG 2 C 条件下 pet-15b-bmp6 pet-32a-bmp6 转化 E. coli BL21(DE3) 表达蛋白的溶解性 Fig. 1 SDS-PAGE analysis of soluble and insoluble protein fractions of pet-15b-bmp6, pet-32a-bmp6 obtained from E. coli BL21(DE3) with.1 mmol/l IPTG at 2 C pmal-p2x-bmp6 E. coli TB1 2 C 37 C, IPTG, 2 C 37 C ; IPTG,.3 mmol/l ( 2, lane 4) 5% SDS-PAGE, MBP-BMP6 ( 3) 图 3 SDS PAGE 分析 pmal-p2x-bmp6 表达融合蛋白 MBP-BMP6 的溶解性 Fig. 3 Analysis of MBP-BMP6 fusion protein expression from the pmal-p2x-bmp6 using SDS PAGE 1: cell extract of uninduced E. coli; 2: cell extract of E. coli after inducing the expression of MBP-BMP6 with IPTG.3 mmol/l at 2 C. 3: insoluble fraction of the E. coli broken with sonication; 4: soluble fraction of sonicated E. coli 图 2 诱导温度和 IPTG 浓度对 pmal-p2x-bmp6/tb1 表达融合蛋白的影响 Fig. 2 Influence of induction temperature, inducer concentration on the expression of soluble MBP-BMP6 from the pmal-p2x-bmp6 in TB1 1: cell extract of uninduced; 2,3 (37 C,IPTG.3 mmol/l,.5 mmol/l): cell extract of E. coli induced; 4,5,6,7(2 C,IPTG.3 mmol/l,.5 mmol/l, 1. mmol/l, 1.5 mmol/l): cell extract of E. coli induced; 8: molecular weight markers pmal-p2x-bmp6 MBP- 图 4.3 mmol/l IPTG 3 C pmal-c2x-bmp6/tb1 诱导表达 MBP-BMP6 融合蛋白 Fig. 4 Expression of the recombinant MBP-BMP6 from the pmal-c2x-bmp6 with.3 mmol/l IPTG at 3 C in TB1 ptwin1-bmp6/er C 3 C 37 C, IPTG. 3 mmol/l,.1 mmol/l 5,

5 456 ISSN1-361 CN /Q Chin J Biotech March 25, 28 Vol.24 No.3, IPTG ; 图 5 诱导温度和 IPTG 浓度对 ptwin1-bmp6/er2566 表达融合蛋白的影响 Fig. 5 Influence of induction temperature, IPTG concentration on the expression of CBD-Intein-BMP6 from the ptwin1-bmp6 in ER2566 1,3 (2 C,IPTG.3 mmol/l,.1 mmol/l): soluble fraction of sonicated cells; 2,4: insoluble fraction; 5,7(3 C, IPTG.3 mmol/l,.1 mmol/l): soluble fraction of sonicated cells; 6, 8: insoluble fraction; 9,11(37 C, IPTG.3 mmol/l,.1 mmol/l): soluble fraction of sonicated cells; 1, 12: insoluble fraction pmal-p2x-bmp6,, pet-32a-bmp6, ptwin1-bmp6,,,,,,,, pmal-p2x-bmp6, 2 C 37 C, pet-15b-bmp6,, 2.2 MBP-BMP6 融合蛋白纯化及 Western blot 鉴定 Amylose, Elution Buffer, 6,, SDS-PAGE 7 表 2 六种融合蛋白在不同诱导条件下表达水平和溶解性比较 Table 2 Expression and solubility levels of rhbmp6 when expressed as six different gene fusions under different induction conditions Recombinant plasmid Temperature / C IPTG concentration /(mmol/l) Expression level Solubility level pgex-5x-bmp pet-15b-bmp6 pet-32a-bmp6 ptwin1-bmp6 pmal-p2x-bmp pmal-c2x-bmp6 3.3 Expression levels given as: =strongest band on SDS-PAGE gel; =among the strongest bands, =visible band. Solubility levels given as: =majority in soluble fraction, =minority in soluble fraction but among the strongest bands, =visible band, =nothing in soluble fraction 6 BMP6 : pmal-c2x-bmp6 图 6 Amylose 亲和纯化 MBP-BMP6 融合蛋白的洗脱图 Fig. 6 Elution profile of MBP-BMP6 fusion protein from Amylase resin with maltose 图 7 Amylose 亲和层析纯化 MBP-BMP6 电泳分析 Fig. 7 Analysis of MBP-BMP6 fusion protein from amylase resin 1: molecular weight markers; 2: soluble fraction of sonicated cells; 3: purified fusion protein from amylose resin

6 : BMP6 457,, Superdex-75 1/3GL ( 8), MBP, SDS-PAGE, 95% ( 9) Western blot 58 kd, MBP- BMP6 图 8 Superdex-75 1/3GL 凝胶层析蛋白洗脱图 Fig. 8 Superdex-75 1/3GL chromatography 1: the purified fusion protein; 2: MBP and degradation products, MBP pmal-c2x-bmp6 XL1-blue TB1 BL21trxB(DE3), MBP-BMP6 MBP-BMP6 XL1-blue, TB1,, Amylose TB1 XL1-blue MBP-BMP6,,, MBP-BMP6 TB1, BL21trx(DE3) BL21trxB(DE3),, BL21trxB(DE3) MBP-BMP6, TB1 ( 1) 图 9 Superdex-75 1/3GL 凝胶层析纯化后 MBP-BMP6 电泳分析及 Western blot 鉴定 Fig. 9 Analysis of final purified fusion protein from Superdex-75 1/3GL chromatography using SDS-PAGE. 1: the affinity column elution contained predominantly MBP-BMP6 fusion protein and MBP; 2: the first peak fraction resulting contained MBP-BMP6 fusion protein; 3: the second peak fraction contained MBP; 4: Western blot analysis of MBP-BMP6 2.3 MBP-BMP6 融合蛋白二聚体的形成及三种 宿主菌表达 MBP-BMP6 的比较 BMP6, BMP6 MBP, 图 1 TB1 和 BL21trxB(DE3) 表达的 MBP-BMP6 亲和纯化后电泳分析 Fig. 1 Soluble fusion protein expressed in TB1 and BL21trxB(DE3)from the affinity column elution. 1: molecular weight markers; 2: fusion protein expressed intotb1; 3: fusion protein expressed in BL21trxB(DE3); 4: molecular weight markers; 5: SDS-PAGE followed by Western blot analysis with an anti-bmp6 of MBP-BMP6 in BL21trxB(DE3) after digesting by Factor Xa 1, TB1 MBP (MW 42.5 kd),, MBP, BL21trxB(DE3) MBP

7 458 ISSN1-361 CN /Q Chin J Biotech March 25, 28 Vol.24 No.3,, TB1, BL21 trxb(de3) TB1 MBP-BMP h SDS-PAGE, ( 11), MBP-BMP6, BL21trxB(DE3), BL21trxB(DE3), TB1 表达的 MBP-BMP6 生物活性的比较, BL21trxB(DE3) MBP-BMP6 C2C12,, 12 ng/ml, TB1,, TB1 图 12 BL21 trxb(de3) 和 TB1 来源 MBP-BMP6 的生物活性比较 Fig. 12 Biological activity of purified MBP-BMP6 from BL21 trxb(de3) and TB1tested by the induction of alkaline phosphatase activity in C2C12 cells 3 讨论 图 11 TB1 来源的融合蛋白稳定性和溶解性分析 Fig. 11 Stability and solubility analysis of fusion protein from TB1 using SDS PAGE. 1: molecular weight marker; 2: fusion protein from the affinity column recently; 3: fusion protein for a week without DTT; 4: fusion protein for a week with DTT, pmal-c2x-bmp6 BL21trxB(DE3) MBP-BMP6, 表 3 2mLpMAL-c2X-BMP6/BL21trxB(DE3) 菌液 MBP-BMP6 纯化效率 Table 3 Purification of the recombinant MBP-BMP6 from the pmal-c2x-bmp6 in BL21trxB(DE3) grown in 2mL LB/AMP Purification step Total Volumn/mL MBP-BMP6 mass/mg Recovery /% MBP-BMP6 purity/% The supernatant after sonication Amylose affinity Superdex-75 1/3GL chromatography , BMPs,,,, ;, ;, 2 DNA, [8 1] BMP6, MBP rhbmp6 GST TRX ;,,,, MBP

8 : BMP6 459,,,,, ;,, MBP, MBP -Seuestered Intermediate MBP,,, MBP,,,,,, BMP6 MBP,, MBP,, ph, ;, [11] pmal-p2x-bmp6, pmal-c2x,,,,, (Thioredoxin ), (Thioredoxin reductase, TrxB) [12] BL21 trxb(de3), rhbmp-6 pmal-c2x-bmp6 BL21 trxb(de3),,,,, pmal-c2x-bmp6 XL1-blue TB1 BL21 trxb (DE3),,,, REFERENCES [1] Urist MR. Bone:formation by autoinduction. Science, 1965, 15(698): [2] Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: Molecular clones and activities. Science, 1988, 242 (4885): [3] Wang EA, Rosen V, Bauduy M, et al. Recombinant human bone morphogenetic protein induces bone formation. Proceedings of the National Academy of Sciences, 199, 87: [4] Gitelman SE, Kobrin MS, Ye JQ, et al. Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo. The Journal of Cell Biology, 1994, 126(6): [5] Gitelman SE, Kirk M, Ye JQ, et al. Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells. Cell Growth & Differentiation, 1995, 6(7): [6] Yang JH, Yang S, Zhao L, et al. Expression and purification rhbmp6 fusion protein in E. coli. Acta Scientiarum Naturalium Universitatis Nankaiensis, 23, 36(1): ,,,. rhbmp-6., 23, 36(1): [7] Yang JH, Zhao L, Yang S, et al. Expression of recombinant human BMP6 in Escherichia coli and its purification and bioassay in vitro. Chinese Journal of Biotechnology, 23, 19(5): ,,,. -6., 23, 19(5): [8] Planson AG, Guijarro JI. Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli. Biochemistry, 23, 42(3): [9] Vallie ER, DiBlasio EA, Kovacic S, et al. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology, 1993, 11(2): [1] Holly EH, John EH. Expression of the class 1 outermembrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag. Protein Expression and Purification, 22, 26(2): [11] De Sutter K, Remaut E, Fiers W. Disulphide bridge formation in the periplasm of Escherichia coli b-lactamase: Human IgG3 hinge fusions as a model system. Molecular Microbiology, 1992, 6(15): [12] Derman AI, Prinz WA, Belin D. Mutations that allow disulfide bond fomation in the cytoplasm of Escherichia coli. Science, 1993, 262(1):