Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.     

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.     

Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1, recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and 0.32 μM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying the biological effects of exogenous rhAng1 in the future.     

Expression, purification and characterization of rat angiopoietin-2 in Pichia pastoris

Angiopoietin-2 (Ang2) is a member of the Ang family. Its potential in clinical use has been highlighted for its important roles in angiogenesis during the individual development and the growth of tumors. Ang2 is difficult to be expressed in E. coli for its unique structure. The expressions of Ang2 in insect cells (Sf9) and Chinese hamster ovary (CHO) cell line have been reported, however, the large-scale production of Ang2 for application is still pendent. In this study, the expression of Ang2 in Pichia pastoris expression system was described for the first time. The cDNA encoding Ang2 was cloned from the rat vascular tissue by RT-PCR, and inserted in the eukaryotic expression vector pPIZαA, and then transformed into P. pastoris KM71H cells. The expression of recombinant rat Ang2 (rrAng2) was induced by methanol and accounted for about 75% of the total secreted proteins. The recombinant protein was subsequently purified by HisTrap FF crude with a purity of 90%. Functional analysis of the purified rrAng2 demonstrated a specific activity in promoting the survival of ECV304 cells and binding to the Tie2 receptor. Preparation of bioactive rrAng2 not only lays the basis for further functional study but also provides a new strategy for soluble and large-scale production of human Ang2.     

Prokaryotic expression,purification and functional characterization of human FHL3

Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42°C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.     

Expression and purification of soluble recombinant Human Endostatin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography. As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.     

Angiogenin Promotes U87MG Cell Proliferation by Activating NF-κB Signaling Pathway and Downregulating Its Binding Partner FHL3

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression.     

转染人核糖核酸酶抑制因子基因对B16黑色瘤细胞及肿瘤转移的影响(英)

人核糖核酸酶抑制因子(human ribonuclease inhibitor, RI)是一种细胞质中分子质量为50 ku的酸性糖蛋白.RI能抑制核糖核酸酶A（RNase A）的活性, RNase A与血管生成因子（angiogenin,Ang）的氨基酸有着高度保守的同源序列.Ang是RNase A超家族的一员,RI通过与RNase A和Ang的紧密结合而抑制其活性.血管生成及新血管的形成, 是肿瘤发生和转移的必要条件.所以抗血管生成将是一种很有希望的对抑制肿瘤生长和转移的有效方法.实验显示RI能有效地抑制肿瘤诱导血管的生成.RI由含有许多亮氨酸重复序列的多肽组成.含有这样重复序列的100多种蛋白质显示了广泛的功能,包括细胞周期调节,DNA修复,对细胞外基质相互作用以及抑制酶活性等.RI被认为是胚胎发育,创伤愈合及肿瘤发生中新血管形成的一种调节因子.RI定位于染色体的11p15.5,与ras基因邻近,在肿瘤病人中经常存在染色体11p15.5部位的变异和异常.RI可能与细胞的生长和分化有关, 因此,RI 可能还具有尚未知的生物学作用.为了进一步了解RI的潜在功能以及探讨RI与肿瘤浸润、转移的关系, 将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317,并转染到B16小鼠黑色瘤细胞中, 用转染空载体和未转染的B16细胞作为对照.通过PCR, RT-PCR, 蛋白质免疫印迹, 免疫荧光分析鉴定,获得稳定表达人核糖核酸酶抑制因子的细胞株.结果显示, 转染的RI基因在体外能显著地抑制细胞增殖和细胞迁移,增加了细胞的粘附以及改善细胞的恶性形态,B16,B16 pLNCX,B16 pLNCX-RI 3种细胞的倍增时间分别为(24.98±0.16) h, (25.62±0.28) h, (32.64±1.11) h.与对照组相比,转RI的细胞粘附率增加17.8%和19.5%而迁移降低了61.4%和60%.转RI的细胞比对照组细胞较平展,核仁和分裂相较少,胞质嗜碱性减弱,提示细胞增殖活性降低和恶性表型的改善. 将3种B16细胞静脉注射到C57BL/6小鼠中, 结果表明, 转染RI基因的实验组显著地抑制了肿瘤的转移, 与两个对照组相比,荷瘤小鼠有更长的存活时间, 少得多的转移节结, 更低的肿瘤血管密度和肺重量.结果显示,RI的表达可能与黑色瘤的转移有关, 提示RI能显著地抑制肿瘤的转移,可能由于其与抑制血管作用,增加细胞粘附,降低细胞迁移及增殖有关.     

Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants

To improve the accumulation of recombinant human epidermal growth factor (hEGF) in transgenic tobacco, a highly effective vector was constructed and transformed via Agrobacterium tumefaciens. The hEGF content in transgenic tobacco was up to 0.3% of the total soluble protein. Using the Vero E6 cell expansion assay and the MTT method for cell proliferation, hEGF produced by transgenic tobacco significantly stimulated Vero E6 cell expansion and proliferation, the same as commercial hEGF products.     

Single chain Fv antibody against angiopoietin-2 inhibits VEGF-induced endothelial cell proliferation and migration in vitro

Angiopoietin-2 (Ang2) promotes tumor growth and metastasis by specifically priming endothelial cells for angiogenesis. Multiple angiogenic factors up-regulate expression of Ang2, suggesting that Ang2 may be the common pathway in growth factor initiated-angiogenesis. Using phage display technology, we generated single chain Fv molecule against human Ang2 (scFv-Ang2) with high affinity (K(d)=0.01 microM) from a mouse phage antibody library. Compared with control scFv, the mouse scFv-Ang2 completely inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) treated with vascular endothelial growth factor (VEGF, 10 ng/ml), but not that of the cells treated with either basic fibroblast growth factor, or angiotensin II, or Ang2. Chemotaxis assay showed that scFv-Ang2 could block completely Ang2-induced (100%) and partially VEGF-induced (49%) migration of HUVECs. The results indicate that Ang2 takes part in the VEGF-induced angiogenesis and scFv-Ang2 might be a promising compound in blocking both VEGF and Ang2 induced angiogenesis.     

Rosiglitzone Suppresses Angiotensin II-Induced Production of KLF5 and Cell Proliferation in Rat Vascular Smooth Muscle Cells

Krüppel-like factor (KLF) 5, which initiates vascular smooth muscle cell (VSMC) proliferation, also participates in Angiotensin (Ang) II-induced vascular remodeling. The protective effect of rosiglitazone on vascular remodeling may be due to their impact on VSMC proliferation. However, the underlying mechanisms involved remain unclear. This study was designed to investigate whether the antiproliferation effects of rosiglitazone are mediated by regulating Ang II/KLF5 response. We found that, in aortas of Ang II-infused rats, vascular remodeling and KLF5 expression were markedly increased, and its target gene cyclin D1 was overexpressed. Co-treatment with rosiglitazone diminished these changes. In growth-arrested VSMCs, PPAR-γ agonists (rosiglitazone and 15d-PGJ2) dose-dependently inhibited Ang II-induced cell proliferation and expression of KLF5 and cyclin D1. Moreover, these effects were attenuated by the PPAR-γ antagonists GW9662, bisphenol A diglycidyl ether and PPAR-γ specific siRNA. Furthermore, rosiglitazone inhibited Ang II-induced phosphorylation of protein kinase C (PKC) ζ and extracellular signal-regulated kinase (ERK) 1/2 and activation of early growth response protein (Egr). In conclusion, in Ang II-stimulated VSMCs, rosiglitazone might have an antiproliferative effect through mechanisms that include reducing KLF5 expression, and a crosstalk between PPAR-γ and PKCζ/ERK_1/2/Egr may be involved in. These findings not only provide a previously unrecognized mechanism by which PPAR-γ agonists inhibit VSMC proliferation, but also document a novel evidence for the beneficial vascular effect of PPAR-γ activation.     

Production of bioactive human hemangiopoietin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95% purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells.     

血管平滑肌细胞增殖与Cdk抑制蛋白p27的表达

ｐ２７蛋白是细胞周期素依赖性激酶（Ｃｄｋ）抑制蛋白家族中的一种,主要对外部促进或抑制细胞增殖的信号起反应。本研究应用流式细胞仪（ＦＣＭ）双标记的方法观察血管紧张素Ⅱ（ＡｎｇⅡ）、血管加压素（ＡＶＰ）和血小板源生长因子（ＰＤＧＦ）对血管平滑肌细胞（ＶＳＭＣｓ）细胞周期百分比和ｐ２７蛋白表达量的影响。静止状态培养的ＶＳＭＣｓ加入ＡｎｇⅡ,ＡＶＰ,ＰＤＧＦＢＢ后,在不同时间收集细胞,用碘化丙啶（ＰＩ）标记细胞ＤＮＡ,以确定细胞所处的周期。用ｐ２７蛋白的单抗和标记了ＦＩＴＣ的二抗标记细胞,通过流式细胞仪测定被激发出的荧光量来确定细胞ｐ２７蛋白表达的相对量。结果显示,ＡｎｇⅡ刺激ＶＳＭＣｓ增生,其蛋白含量增加了４３６％（Ｐ＜００１）,但不抑制ｐ２７蛋白的表达;ＡＶＰ可轻度抑制ｐ２７的表达,有轻度促进ＶＳＭＣｓ增殖和增生的作用（Ｐ＜００５）;ＰＤＧＦ明显抑制ｐ２７的表达,引起细胞增殖。本研究结果提示,ｐ２７蛋白抑制ＶＳＭＣｓ通过Ｇ１期进入Ｓ期,是抑制ＶＳＭＣｓ增殖的重要调节因子。     

人骨骼肌肌钙蛋白C在大肠杆菌中的表达及其抗肿瘤作用

为了探索重组人骨骼肌型肌钙蛋白C(rhTnC)的抗肿瘤作用,采用PCR技术,从人乳腺cDNA文库中获得编码TnC的cDNA序列,在大肠杆菌中进行了表达.经镍柱亲和层析纯化后,分别检测其在体外对人脐静脉内皮细胞生长的抑制作用与对小鼠异位移植肿瘤(S-180)生长的抑制作用.结果表明,rhTnC在大肠杆菌中呈可溶性表达.分离纯化后的rhTnC在体外对人脐静脉内皮细胞生长呈剂量依赖性抑制(IC50为7.5μg/ml);对小鼠异位移植肿瘤的生长也有明显的抑制作用,在20mg/(kg·d)剂量下对S-180的抑制率为64.4%.以上结果说明,rhTnC可抑制血管内皮细胞生长,并具有抗肿瘤作用.     

Association of Angiopoietin-2 and Ki-67 Expression with Vascular Density and Sunitinib Response in Metastatic Renal Cell Carcinoma

The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS.     

High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO–scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO–scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.     

Transient expression, purification and characterization of bioactive human fibroblast growth factor 8b in tobacco plants

cDNA of human fibroblast growth factor 8 isoform b (FGF8b) was cloned for the first time into a plant expression vector with or without endoplasmic reticulum retention signal (KDEL) and was transiently expressed as His tagged fusion protein in Nicotiana tabacum leaves through Agrobacterium mediated gene transfer by vacuum infiltration method. Expression of FGF8b was confirmed by ELISA and Western blot using anti-FGF8b antibody and the expression level was measured as 4.1% of total soluble protein of tobacco leaves. The expressed recombinant protein was purified by Ni-NTA affinity chromatography and its molecular weight was determined by MALDI-TOF-MS. Schiff’s test, Concanavalin A (Con A) immunoblot and enzymatic deglycosylation indicated that the high molecular mass was due to glycosylation of the FGF8b expressed in plant cells. Measurement of its biological activity in NIH3T3 cells by thymidine incorporation and MTT assay showed induction of cell proliferation. These results indicate that biologically active recombinant FGF8b could be expressed in tobacco plants. Surya Kumar Potula and Sonal Roy Kathuria contributed equally to this work.     

Automatic laboratory-scale fed-batch procedure for production of recombinant proteins using inducible expression systems of Escherichia coli

Summary An automatic fed-batch procedure for the production of recombinant proteins in Escherichia coli was developed. Using glycerol as carbon source and by controlling the growth rate by using feed-forward algorithm, enabled high specific expression level (10–20 % of total cell protein) at high cell densities (20 g dry wt/l) to be achieved: rat and human soluble catechol-O-methyltransferase, calf prochymosin, and human troponin C were expressed with nearly 50-fold higher volumetric yield compared to the conventional (batch) procedures.     

Aptamers improve the expression of a human granulocyte-macrophage colony stimulating factor in transgenicArabidopsis thaliana seeds

Domain IV nucleotides of human 7SL RNA comprise an approximately 50-nucleotide region with a highly conserved secondary structure consisting of two internal loops. We inserted these into a modified 3’-untranslated region of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene. The expression vector, under the control of a tissue-specific promoter derived from the soybean α’ subunit of β-conglycinin, was introduced intoArabidopsis thaliana byAgrohacterium-mediated transformation. Expression of the recombinant hGM-CSF significantly improved, accounting for as much as 0.049% of the total soluble protein in theArabidopsis siliques. Western blots showed that the molecular weight of this recombinant hGM-CSF was approximately 21 kD. In addition, TF-1 cell proliferation data demonstrated that the recombinant hGM-CSF was biologically active. Our results provide evidence that aptamers can strongly enhance gene expression.