A novel expression system for recombinant marine mussel adhesive protein Mefp1 using a truncated OmpA signal peptide

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (> or = 10.55). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP (OmpASP(tr)) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with OmpASP(tr) peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an OmpASP(tr)| Xa leader sequence that contains the recognition site for Xa protease.     

Novel GFP expression using a short N-terminal polypeptide through the defined twin-arginine translocation (Tat) pathway

Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔGRNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.     

Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.

The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.     

水稻白叶枯病抗性基因Xα21和稻瘟病抗性基因Pi-d2激酶蛋白质在毕赤酵母中的表达及条件优化

[目的]白叶枯病和稻瘟病是最主要的水稻病害,Xα21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质.本研究旨在毕赤酵母系统中表达XA21和PI-D2激酶蛋白质.[方法]用Xα21和Pi-d2的激酶区PCR产物,构建了pPICZαA-Xα21K、pPICZαA-Pi-d2K重组质粒,酶切及测序验证后,将重组质粒线性化,转化到毕赤酵母菌株中,系统地比较了不同酵母菌株(KM71、GS115、X33),不同甲醇浓度(1%、2%、3%),不同pH(pH5、pH6、pH7、pH8)值,不同诱导时间(24 h、48 h、72 h)条件下激酶蛋白质的表达情况.[结果]XA21和PI-D2激酶蛋白质可以在毕赤酵母中表达,但表达的蛋白质不能分泌到培养基上清中,而只能在菌体中检测到,对表达条件的系统比较发现,毕赤酵母菌株KM71和X33、2%的甲醇诱导浓度、pH5和48 h以上的诱导时间有利于激酶蛋白质的表达,最后我们在酵母裂解物上清中获得了纯化的考染可见的激酶蛋白质.[结论]在毕赤酵母中表达了XA21和PI-D2激酶蛋白质,为下一步生化特性研究奠定了基础.     

Expression of human plasminogen kringle 5 as fusion protein with truncated hIFNgamma gene in Escherichia coli

The human interferon gamma (hIFNgamma) gene was used as a fusion partner to mediate the expression of heterologous proteins and the effect of the fusion partner length on the expression of the heterologous protein was researched. Plasminogen kringle 5 (pk5), an inhibitor of angiogenesis, was fused to hIFNgamma and its serially truncated fragments, respectively, and the expression of fusion proteins was determined by SDS-Page gel. The pk5 protein was obtained readily by the introduction of sequences recognized by protease factor Xa at the fusion site and ion-exchange chromatography was employed to purify pk5. The recovery of the biological activities of pk5 was studied using the orthogonal experimental design L9 (3(4)) (four factors, three levels, nine experiments) and evaluated by measurement of anti-endothelial cell proliferation in vitro.     

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.     

蛇神经毒素的表达和鉴定

抽提中华眼镜蛇毒腺总RNA,通过反转录PCR扩增Cobrotoxin cDNA,克隆并测序。该cDNA编码83个氨基酸,包括21个氨基酸的信号肽和62个氨基酸的成熟蛋白。该成熟蛋白的氨基酸序列和通过蛋白测序从台湾眼镜蛇鉴定的Cobrotoxin完全一致。PCR扩增编码Cobrotoxin的DNA,并亚克隆到表达载体pMAL-P₂。此外,通过合成寡核苷酸片段,拼接成完整的CM11基因,并将其克隆至pMAL-P₂。经IPTG诱导,两种神经毒素基因在大肠杆菌中都得到高效的可溶性融合表达。表达产物通过SDS-PAGE和蛋白印迹杂交加以鉴定。表达的融合蛋白经过Sepharose 6Bamylose亲和色谱和DEAESepharose FF离子交换色谱得到有效纯化。经Xa因子酶切后得到的两种重组神经毒素都具有小白鼠体内毒性。     

A set of cassettes and improved vectors for genetic and biochemical characterization of Pseudomonas genes

A set of broad-host-range vectors allowing direct selection of recombinant DNA molecules to facilitate subcloning and expression analyses of Pseudomonas genes was constructed using Bg/II lacZ alpha cassette. Controlled expression vectors pVDtac39 and pVDtac24 were shown to be useful for determination of enzymatic activities encoded by the cloned DNA fragments and Mr determination of the corresponding polypeptides. A set of Pseudomonas putida xylE gene cassettes truncated at the 5' end was constructed for translational (protein) fusion studies. A protein fusion of the Pseudomonas aeruginosa algD gene, coding for GDPmannose dehydrogenase, and the truncated xylE gene cassette was used to verify the putative coding region and translational signals predicted from the algD nucleotide sequence.     

Production of extracellular domain of human tissue factor using maltose-binding protein fusion system

Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases. Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available. In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli. The coding region of extracellular domain of tissue factor was linked to the 3(')-end of maltose-binding protein (MBP) gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tTF was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 21.5% of the total soluble protein in E. coli, allowing approximately 15mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay. This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF.     

蛋白A信号肽引导的E.coli外泌高表达异源蛋白

利用葡萄球菌ｐｒｏｔｅｉｎ Ａ信号序列（ＳＰＡ）,我们构建了不同启动子控制的分泌表达质粒。经表达研究,获得了可控性好、表达量高的ＰＬ启动子控制的分泌表达载体。通过菌种的筛选、培养条件和诱导条件的摸索,获得了能将表达产物的绝大部分分泌到培养液中的大肠杆菌高效外泌表达系统,外泌表达量可达１００ｍｇ／Ｌ（菌浓度为１Ａ６００／ｍｌ）以上,如此高的分泌表达量尚未见文献报道。利用该系统成功、高效地外泌表达了ｐ     

重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性     

The solubility and stability of recombinant proteins are increased by their fusion to NusA

The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.     

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.     

Accurate disulfide formation in Escherichia coli: overexpression and characterization of the first domain (HF6478) of the multiple Kazal-type inhibitor LEKTI

The human hemofiltrate peptide HF6478, a putative serine proteinase inhibitor, which is part of the precursor protein LEKTI, was cloned, overexpressed, and purified. HF6478 contains two disulfide bridges with 1-4, 2-3 connectivity, sharing partial homology to Kazal-type domains and other serine proteinase inhibitors. It was expressed as thioredoxin (Trx) fusion protein, and disulfide formation occurred in the oxidative cytoplasm of Escherichia coli Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation. The soluble fusion protein was purified using metal-chelating affinity chromatography. Cleavage of the Trx fusion protein with factor Xa and subsequent purification yielded the final product in amounts sufficient for structural studies. Characterization of recombinant HF6478 was done by amino acid sequencing, mass spectrometry, capillary zone electrophoresis, and CD spectroscopy. Taking the blood filtrate peptide HF6478 as example, we present a strategy which should facilitate the expression of different extracellular proteins in the E. coli cytoplasm.     

Systematic high-yield production of human secreted proteins in Escherichia coli

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.     

Expression cloned cDNA for 10-deacetylbaccatin III-10-O-acetyltransferase in Escherichia coli: a comparative study of three fusion systems

10-Deacetylbaccatin III-10-O-acetyltransferase (10-DABT) catalyzes the formation of baccatin III, which is an immediate diterpenoid precursor of Taxol. A cDNA encoding 10-DABT was cloned from Taxus baccata by using RT-PCR and screening a cDNA library. A study of its heterologous overexpression in Escherichia coli was carried out. To get high-level expression of recombinant enzyme, three kinds of IPTG inducible fusion expression systems (with glutathione S-transferase (GST), hexahistidine (6x His), and biotinylated tag) were used, and results of expression were compared. Fusion 10-DABT with different tags was expressed with diverse expression levels and solubility in the three systems. Optimum IPTG concentration, temperature, and inducing time for producing recombinant enzymes were found. Under higher IPTG concentration (up to 1 mM), the highest level of expression for fusion protein was obtained in the 6x His fusion system with phage T5 promoter, but expressed products were only partially soluble. With lower IPTG concentration (less than 0.5 mM), the highest expression was detected in the GST fusion system with tac promoter, and the lowest level of expression appeared in the biotinylated fusion system. The expression level in the latter system did not differ dramatically with a range of different inducer concentrations. GST and 6x His fusion proteins were mainly soluble in aqueous solutions and Triton X-100 improved the solubility of biotinylated fusion proteins (inferring this protein is membrane-associated). Fusion proteins could only be partially purified by a single affinity chromatography step for all three systems. Glutathione-coupled matrix and streptavidin-conjugated resin have higher specificity than Ni-NTA resin, and elution conditions were shown to affect enzyme activity. Three kinds of recombinant 10-DABT with different tags showed enzyme activity, but total enzyme activity was lost as a result of the affinity chromatography step. Thrombin and Factor Xa could be used for site-specific cleavage of fusion proteins, but the incubation temperature affected enzyme activity of recombinant enzymes.     

提高大肠杆菌可溶性重组蛋白表达产率的研究进展

大肠杆菌表达重组蛋白相比真核细胞具有成本低廉、大规模发酵容易、条件易于自动化控制等优点,通过大肠杆菌表达重组蛋白是一种高效、经济的途径,重组蛋白表达量可达到大肠杆菌总蛋白质量的50%。具有正常生化活性的重组蛋白通常为可溶性形式,因而对于以得到活性产物(如抗体、酶等)为目的的研究,通常采用可溶性表达途径。目前已有多种以可溶性重组蛋白为活性物质的治疗性药物经批准上市,但并非所有外源基因均能实现可溶性高表达,因此重组蛋白的可溶性高表达具有重要研究价值。在总结近年提高经大肠杆菌可溶性表达重组蛋白产率研究的基础上,从启动子的选择、SD序列的引入、信号肽的优化、宿主细胞的选择、共表达其他蛋白质,高密度发酵等方面阐释在大肠杆菌中提高可溶性重组蛋白表达产率的方法。     

N端融合不同长度转位肽的重组粒酶B抑制细胞生长作用的比较

采用重组PCR法将粒酶B基因的N端信号肽和酸性二肽编码序列去除,与两种不同长度的绿脓杆菌外毒素（PE）转位肽序列分别连接,将它们插入pIND诱导表达载体,通过脂质体法与pVgRXR辅助质粒共转染HeLa细胞,建立了重组PE II-GrBa基因的诱导表达细胞系。松甾酮A诱导后Western印迹检测到目的基因的表达,间接免疫荧光观察到表达细胞出现多核巨细胞的异常形态。两种表达的PE II-GrBa融合蛋白均能够切割粒酶B的细胞内源性和外源性底物,并且使细胞生长速度减慢。其中,PE II-(aa 280358)-GrBa的底物切割能力和生长抑制作用较强。流式细胞仪分析这种抑制作用可能与细胞周期的G2期受到阻遏有关。上述结果证实了PE II-GrBa融合蛋白仍然具有抑制细胞生长的作用,并且较短的转位肽对GrBa活性的影响较小,有助于进一步优化转位肽/细胞毒性效应蛋白重组分子的结构用于肿瘤细胞杀伤。     

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.     

Expression of honeybee prepromelittin as a fusion protein in Escherichia coli.

Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.