重组FAS分子的表达、纯化及抗体制备

用ＰＣＲ法扩增出编码人ＦＡＳ分子胞外区的ｃＤＮＡ片段,直接克隆到ｐＧＥＭ－Ｔ载体上,经ＤＮＡ序列测定后,再插入到谷胱甘肽转硫酶（ＧＳＴ）融合蛋白表达载体ｐＧＥＸ－ＫＧ的ＥｃｏＲⅠ和ＳａｌⅠ位点之间,构成重组质粒ｐＫＧ－ｈＦＡＳ,将此质粒导入大肠杆菌,经ＩＰＴＧ诱导后获得ＧＳＴ－ｈＦＡＳ重组融合蛋白的表达,用谷胱甘肽偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ经亲合层析获得纯化的ＧＳＴ－ｈＦＡＳ蛋白,经凝血酶酶切和二次亲合层析去除ＧＳＴ部分,得到纯化的ＦＡＳ蛋白．用纯化的ＦＡＳ抗原免疫家兔制备了抗ＦＡＳ抗体,经检测发现抗ＦＡＳ抗体能诱导Ｕ９３７细胞发生细胞凋亡     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

缩短的人巨噬细胞集落刺激因子（M—CSF）在大肠杆菌中…

本文用聚合酶链反应（ＰＣＲ）获得了一个缩短的人巨噬细胞集落刺激因子ｃＤＮＡ基因,并克隆在质粒ｐＡＥＴ３ｄ中,在Ｔ７启动子指导下,在大肠杆菌ＢＬ２ＬｙｓＥ中获得了和一个６组氨酸短肽标签的融合表达。重组的融合Ｍ－ＣＳＦ表达量占菌体总蛋白的１２％,表达产物一部分以不溶性包涵体形式存在,另一部分则以可溶性蛋白存在。经过金属螯合新和层析一步纯化,所得的融合（Ｈｉｓ）６－Ｍ－ＣＳＦ在还原型ＳＤＳ－ＰＡＧＥ上基     

蓝细菌Synechococcus sp.PCC7002petH基因的高效表达及其产物的纯化

铁氧还原白－ＮＡＤＰ＾＋氧还酶（ＦＮＲ）是光合电子传递中的关键酶之一。蓝细菌Ｓｙｎｅｃｈｏｃｏｃｃｕｓ ｓｐ．ＰＣＣ７００２中编码ＦＮＲ的ｐｅｔＨ基因被克隆到表达载体ｐＥＴ－３ｄ上,在大肠杆菌中得到了高效表达,其表达量占大肠杆菌总蛋白的５０５％以上。高效表达的产物以可溶性和包含体两种形式存在。可溶性部分具有ＦＮＲ的酶活性,在经过离子交换和凝胶层析后产生了电泳纯的ＦＮＲ。包含体形式的部分经６．７ｍｏ     

人表皮生长因子(EGF)与单核-巨噬细胞集落刺激因子(GM-CSF)融合蛋白在大肠杆菌中的表达

应用基因工程技术,将ＥＧＦ、ＧＭ－ＣＳＦ基因克隆到ｐＧＥＭ－３Ｚｆ（＋）载体的ＥｃｏＲＩ,ＢａｍＨＩ位点上,再将重组融合基因亚克隆到表达载体ｐＢＶ２２０的ＥｃｏＲＩ,ＢａｍＨＩ位点上,在大肠杆菌ＤＨ５α中进行表达,ＳＤＳ－聚丙烯酰胺凝胶电泳和Ｗｅｓｔｅｒｎｂｌｏｔ表明ＥＧＦ－ＧＭ－ＣＳＦ融合蛋白获得表达,并且具有ＥＧＦ、ＧＭ－ＣＳＦ的免疫学活性．这为进一步研究该融合蛋白的功能和肿瘤治疗提供一种新的基因产品．     

人表皮生长因子（EGF）与单核—巨噬细胞集落刺激因子（GM—CSF）融合？…

应用基因工程技术,将ＥＧＦ,ＧＭ－ＣＳＦ基因克隆到ｐＧＥＭ－３Ｚｆ载体的ＥｃｏＲＩ,ＢａｍＨＩ位点上,再钭重组融合基因亚克隆到表达载体ｐＢＶ２２０扔ＥｃｏＲＩ,ＢａｍＨＩ位点上,在大肠杆菌ＤＨ５α中进行表达,ＳＤＳ－聚丙烯酰胺凝胶电泳和Ｗｅｓｔｅｒｎ ｂｌｏｔ表明ＥＧ－ＦＧＭ－ＣＳＦ融全蛋白获得表达,并且具有ＥＧＦ,ＧＭ－ＣＳＦ免疫学活活。     

人GM—CSF cDNA的克隆和在大肠杆菌中的表达

从诱导的人胚肺细胞ＨＦＬ株中提取总ＲＮＡ．经ＲＴ－ＰＣＲ反应获取了人ＧＭ－ＣＳＦｃＤＮＡ,ＤＮＡ序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用ＰＣＲ改造了人ＧＭ－ＣＳＦ的ｃＤＮＡ５’端核苷酸序列,并将改造的人ＧＭ－ＣＳＦ基因插入含Ｔ７启动子的质粒ｐＥＴ－１１ｄ构建成表达质粒ｐＥＴＣ－５,将此质粒转化大肠杆菌株ＢＬ２１（ＤＥ３）得到表达菌株ＢＬＥＣ４。表达菌株用０．５ｍｏｌ／ＬＩＰＴＧ诱导２小时后,产生大量重组蛋白并形成包涵体。ＳＤＳ—ＰＡＧＥ电泳图谱扫描结果表明,ｒｈＧＭ－ＣＳＦ产量占菌体总蛋白量的１６％。ＥＬＩＳＡ和ＴＦ－１细胞培养测定表明,初步纯化和复性的ｒｈＧＭ－ＣＳＦ具有天然的ｈＧＭ－ＣＳＦ生物活性。     

重组人MCAF／GM－CSF融合蛋白表达及其生物学活性研究

利用ＰＣＲ技术和ＤＮＡ体外重组方法,把作为导向效应细胞到靶部位的单核细胞趋化激活因子（ＭＣＡＦ）和粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）进行基因融合,置于ｐＢＶ２２０载体的λＰＲＰＬ串联启动子下游,构建了ＳＤ序列与ＡＴＧ之间含有不同核苷酸组成的重组质粒ｐＭＧ０１、ｐＭＧ０２和ｐＭＧ０３。ｐＭＧ０１、ｐＭＧ０２和ｐＭＧ０３的翻译起始区都不存在稳定的二级结构,但ＤＨ５α（ｐＭＧ０２、ＤＨ５α（ｐＭＧ０３）的表达水平远远高于ＤＨ５α（ｐＭＧ０１）,ＤＨ５α（ＰＭＧ０１）几乎没有表达。表达产物经Ｗｅｓｔｅｒｎｂｌｏｔ检测表明,它能分别与ＭＣＡＦ和ＧＭ－ＣＳＦ抗体发生特异反应。生物学活性测定表明,表达产物具有明显的单核细胞趋化活性和维持ｈＧＭ－ＣＳＦ依赖的ＴＦ１细胞生长的特性,说明ＭＣＡＦ和ＧＭ－ＣＳＦ的生物学功能是相容的．     

重组人GM—CSF基因在昆虫细胞中的表达

利用苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）带β－Ｇａｌａｃｔｏｓｉｄａｓｅ基因标记的非融合蛋白基因转移载体ｐＢｌｕｅＢａｃ将人粒细胞巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）基因成功地插入病毒ＡｃＮＰＶ的基因组中．ｈＧＭ－ＣＳＦ基因在感染重组病毒的草地夜蛾（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）培养细胞Ｓｆ９中得到表达,感染后的Ｓｆ９细胞培养液能刺激人骨髓细胞在体外形成典型的集落,表达水平可达２．７×１０５５ＣＦＵ／ｍｌ。以ｈＧＭ－ＣＳＦ单抗所作的ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ表明,表达的ｈＧＭ－ＣＳＦ对是３种糖基化程度不同的产物,分子量分别约为１５ｋｄ,１８ｋｄ和２０ｋｄ。     

凝血酶激活的单链尿激酶型纤溶酶原激活剂的构建、表达、纯化和性质研究

用Ｋｕｎｋｅｌ突变法,将单链尿激酶型纤溶酶原激活剂（ｓｃｕ－ＰＡ）ｃＤＮＡ基因中编码Ｐｒｏ１５５—Ｌｙｓ１５８的片段定点突变,并将此突变的ｓｃｕ－ＰＡ（ｔｓｃｕ－ＰＡ）的ｃＤＮＡ克隆到表达载体ｐＣＭ－β－ｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞．获得的稳定表达株在无血清培养基中２４ｈ的表达量为６２０ＩＵ／１０６细胞．经锌离子螯合Ｓｅｐｈａｒｏｓｅ亲和层析得到ｔｓｃｕ－ＰＡ纯品．ＳＤＳ－ＰＡＧＥ显示ｔｓｃｕ－ＰＡ分子量为５３ｋＤ左右,与预期的结果相符．ｔｓｃｕ－ＰＡ是由凝血酶激活而不是由纤溶酶激活,但激活后也能转变为双链分子（ｔｃｕ－ＰＡ）．ｔｓｃｕ－ＰＡ仍保持了ｓｃｕ－ＰＡ的血纤维蛋白亲和性．酶动力学研究表明,激活后的ｔｓｃｕ－ＰＡ水解Ｓ２４４４的Ｋｍ和Ｋｃａｔ值与高分子量尿激酶（ＨＵＫ）相似．体外溶栓实验结果表明,ｔｓｃｕ－ＰＡ可以选择性地溶解富含凝血酶的血凝块,对贫凝血酶的血凝块作用不大     

人内皮抑素基因的克隆以及在大肠杆菌中的可溶性表达研究

用PCR的方法从人胎肝cDNA文库中得到人内皮抑素基因 ,克隆测序正确后连接到硫氧还蛋白融合表达载体上 ,转化大肠杆菌BL2 1 (DE3)得到表达人内皮抑素的工程菌。用IPTG诱导表达 ,表达量达到全菌蛋白的 64%。经分析硫氧还蛋白可以辅助内皮抑素可溶性表达 ,表达的融合蛋白保持了天然蛋白的免疫学特性。而且表面带有多聚组氨酸的突变的硫氧还蛋白还简化了蛋白纯化的步骤 ,使融合蛋白可以通过固相金属螯和层析 (IMAC)的方法纯化。纯化后的融合蛋白经IgA蛋白酶的切割可得到大小正确的重组人内皮抑素 ,用此方法获得的重组人内皮抑素可以在CAM试验中抑制新生血管的形成。高效可溶型表达内皮抑素的工程菌的构建成功 ,为内皮抑素的生产应用打下了良好的基础。     

Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli

Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium). Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography. The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A. Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas.     

New fusion protein systems designed to give soluble expression in Escherichia coli.

Three native E. coli proteins-NusA, GrpE, and bacterioferritin (BFR)-were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37 degrees C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37 degrees C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-gamma were also expressed in E. coli at 37 degrees C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography.     

A unique approach for high level expression and production of a recombinant cobra neurotoxin in Escherichia coli

In this report, we describe a simple approach to produce a large quantity of a recombinant cobra neurotoxin containing four pairs of disulfide bonds. A cDNA encoding the toxin was fused, in frame, to the carboxyl termini of thioredoxin via a linker sequence encoding two amino acids, Asp and Pro. Due to the presence of thioredoxin, a soluble form of the fusion protein was expressed in a compartment, sensitive to osmotic pressure, in Escherichia coli. The fusion protein was released into the solution with low ionic strength under an osmotic shock treatment, and purified in a single step using an ion exchange chromatography column. The purified protein was treated in diluted hydrochloric acid to induce hydrolysis of the protein at the Asp-Pro linker site. Then, the recombinant neurotoxin was purified by gel filtration of the acid-treated sample. When the biological activity of the purified toxin was assayed, it was as potent as the natural toxin. Using this protocol, approximately 12 mg of pure recombinant neurotoxin can be produced from one liter of bacterial culture. More importantly, this protocol can be easily used for the production of the toxin at a larger scale with low cost. The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.     

Synthesis of 32P-labelled protein probes using a modified thioredoxin fusion protein expression system in Escherichia coli

The thioredoxin fusion protein expression system from invitrogen was modified so that 32P-labelled recombinant proteins can be easily obtained in large quantities for functional studies. Proteins that are prone to form the inclusion bodies can be functionally expressed as thioredoxin fusion proteins in Escherichia coli. After expression, the recombinant proteins can be easily phosphorylated with 32P-gamma ATP and the 32P-labelled protein can be obtained functionally via a mild proteolytic digestion to cleave off the thioredoxin moiety. A deletion construct of the Ah receptor nuclear translocator protein was used as an example to illustrate how this protein expression system works.     

Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2(t), residues 196-464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2(t) production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP-recROP2(t) being readily expressed in a soluble form. Moreover, the insoluble form of MBP-recROP2(t) could be correctly refolded with a recovery of more than 80%. Both forms of MBP-recROP2(t) were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX-recROP2(t) promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX-recROP2(t) and of MBP-recROP2(t) led respectively to the complete degradation or to the truncation of the recROP2(t) moiety. However, recROP2(t), despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.     

Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein αi1

Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigation of intramolecular motions in RGS and Gα proteins in their apo and complexed forms, we have successfully developed a protocol for preparing milligram quantities of highly purified, isotopically labeled wild-type human Gα(i1) (hGα(i1)) subunit for NMR studies. High levels of expression in Escherichia coli can be attributed to the use of the SUMO fusion protein system, a bacterial strain that produces rare codons, supplementation of minimal medium with small quantities of isotopically labeled rich medium and a lowered induction temperature. Purification of hGα(i1) utilized affinity and size exclusion chromatography, and protein activity was confirmed using fluorescence-based GTP-binding studies. Preliminary NMR analysis of hGα(i1) has shown that high-quality spectra can be obtained at near-physiological temperatures, whereas lower temperature spectra display numerous weak and broadened peaks, providing preliminary evidence for widespread μs-ms timescale exchange. In an effort to further optimize the NMR spectra we prepared a truncated form of hGα(i1) (hGα(i1)-Δ31) in which the 31-residue unstructured N-terminus was removed. This resulted in further improvements in spectral quality by eliminating high-intensity peaks that obscured resonances from structured segments of the protein. We plan to use hGα(i1)-Δ31 in future investigations of protein dynamics by NMR spectroscopy to gain insight into the role of these motions in RGS/Gα binding selectivity.     

Expression of human cardiac-specific homeobox protein in Escherichia coli

Human cardiac-specific homeobox protein cDNA (hCsx) was cloned into expression plasmid pET32a and fused with Escherichia coli thioredoxin (Trx). The Trx-Csx fusion protein was under the control of bacteriophage T7 promoter. When expressed in E. coli BL21(DE3), about half of the recombinant Trx-Csx products existed in the form of insoluble inclusion bodies. When coexpressed with human protein disulfide isomerase, more than 90% of Trx-Csx products accumulated in the soluble form in the cell lysate. The recombinant Csx fusion protein was purified by one-step metal-chelating affinity chromatography.     

Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites

C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited internally replacing the DDDDK in another construct of thioredoxin/mouse NT-proCNP, it was found to be poorly processed by enterokinase. Our results showed that non-target sequences can be preferentially recognized over the canonical DDDDK sequence when located accessibly at the ends of proteins.     

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.