Mac-1-FP融合表达载体的构建、Mac-1-FP的表达与活性鉴定

分别构建表达BFP与CD1 1b的C末端、YFP与CD1 8的N末端相连接的融合蛋白的表达载体 ,并将二者转染至既无内源性Mac 1的表达同时又具有某些炎症反应信号转导系统的CHO细胞株进行表达Mac 1 FP .通过荧光显微镜观察到共转染后的CHO细胞可发出蓝色荧光和黄色荧光 ,应用Western印迹方法确定CD1 1b BFP与YFP CD1 8能够形成二聚体 ,采用流式细胞术检测确定PMA刺激Mac 1 FP可由胞浆内转位至膜上 ,测定PMA刺激前后的转染CHO细胞与其配基ICAM 1粘附活性的变化 ,证明转染CHO中的Mac 1 FP表达成功并具有野生型Mac 1的形成二聚体、膜转位、和配基ICAM 1相结合等功能 ,为进一步研究白细胞表面粘附分子Mac 1的α亚基CD1 1b、β亚基CD1 8在细胞内的走向及归宿创造了条件     

A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells

We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.     

Green fluorescent antibodies: novel in vitro tools

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.     

Engineering mRNA translation initiation to enhance transient gene expression in chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser(51)Ala site-directed mutant of eIF2alpha, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2alpha by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single- and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2alpha Ser(51)Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser(51)Ala or wild-type eIF2alpha proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.     

Using storage organelles for the accumulation and encapsulation of recombinant proteins

Plants have been used to produce many diverse and valuable recombinant proteins, including subunit vaccines, antibodies and antibody fragments, hormones, blood products, cytokines, and enzymes. Different plant species and platforms have been explored as production hosts, each with unique properties in terms of the gene transfer method, production time, environmental containment, scalability, downstream processing strategy, protein folding and accumulation, and overall costs. Seed-based systems have many advantages because they exploit the natural storage properties of seeds, which facilitate batch processing and distribution. Seeds possess specialized storage organelles that may be used to accumulate recombinant proteins, offering stability both in planta and after harvest in the final preparation/formulation. The post-harvest stabilizing effect of seeds allows recombinant subunit vaccines and antibodies to be delivered via the mucosal route because they are better able to withstand this harsh microenvironment when protected by the plant matrix. Native storage organelles such as starch granules and protein bodies offer this protective effect, but protein storage organelles can also be induced ectopically in vegetative tissues. In this paper, we discuss the technical capabilities of storage organelle-based expression platforms and their potential applications.     

High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.     

Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories. Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3. The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity. The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence. The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3. Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3. Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.     

Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture

A platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.     

人尿激酶型纤溶酶原激活因子截短型突变体与绿色荧光蛋白在真核细胞HEK293F中的融合表达

目的:构建人尿激酶型纤溶酶原激活因子(uPA)截短型突变体与绿色荧光蛋白(EGFP)分泌型融合表达载体并在真核细胞中表达。方法:采用PCR法,分别以质粒pIRES2-EGFP和重组质粒pcDNA3.1(+)/uPA为模板,扩增出带BamHⅠ和XbaⅠ酶切位点的EGFP及带NheⅠ和HindⅢ酶切位点的uPA截短体基因片段,先后将EGFP和截短型uPA基因片段克隆到真核表达载体pcDNA3.1(+)上,转入HEK293F细胞,用G418对转染细胞进行加压筛选,通过共聚焦显微镜观察和ELISA方法鉴定表达产物。结果:DNA测序结果显示,uPA不同截短型突变体基因片段与EGFP基因融合的真核表达载体构建成功,共聚焦显微镜观察发现HEK293F细胞中有绿色荧光且定位于细胞质中,ELISA检测到HEK293F细胞培养上清中分泌型融合蛋白的表达。结论:构建了uPA截短型突变体与EGFP分泌型融合表达载体并在真核细胞中表达,为后期研究uPA的相互作用蛋白及其生理功能奠定了基础。     

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

Background  

Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct.     

杜氏盐藻核基质结合区结合蛋白的细胞定位

为研究核基质结合区结合蛋白的功能及调控机制,PCR扩增杜氏盐藻MBP的cDNA全长序列及N端和C端序列,与绿色荧光蛋白基因融合构建真核表达载体,脂质体转染CHO细胞,Western blotting和荧光显微镜检测基因表达情况和细胞定位。结果显示:MBP及N端和C端融合蛋白成功在CHO细胞表达,MBP和C端部分定位于细胞核且聚集于核仁,N端部分分布整个细胞,说明MBP定位于细胞核且细胞定位信号位于C端,MBP可能与rRNA前体结合发挥作用。     

Expression of double foreign protein types following recombinant baculovirus infection of stably transfected Drosophila S2 cells

We sought to develop a platform for simultaneous, regulatable expression of double foreign protein types in cell culture. Drosophila melanogaster Schneider line 2 (S2) insect cells that stably express human erythropoietin (hEPO) were infected with a recombinant baculovirus containing the green fluorescent protein (GFP) gene. Since baculovirus cannot replicate in nonpermissive S2 cells, baculovirus infection did not affect cell growth or viability. Expression of each foreign protein was under the control of the inducible metallothionein (MT) promoter. Addition of copper sulfate to infected, stably transfected cells resulted in simultaneous expression of both GFP and hEPO. Induced hEPO expression profile and levels were similar in both control and infected cells, indicating that baculovirus infection also did not affect expression of stably introduced foreign gene. GFP protein levels were regulated by the infection dose of recombinant baculovirus, while hEPO expression remained constant. hEPO levels were much higher (30-fold) than GFP, indicating plasmid-based introduced gene copies have higher expression than baculovirus-based introduced genes. These data suggest the baculovirus/stable S2 cell system can be used to produce a major target protein by plasmid-based stable transfection, and assistant proteins by recombinant baculovirus infection. Such a system appears to be very attractive as a multiple protein expression platform for engineering metabolic pathways in cell culture.     

SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins

SUMO (small ubiquitin-related modifier) modulates protein structure and function by covalently binding to the lysine side chains of the target proteins. Yeast cells contain two SUMO proteases, Ulp1 and Ulp2, that cleave sumoylated proteins in the cell. Ulp1 (SUMO protease 1) processes the SUMO precursor to its mature form and also de-conjugates SUMO from side chain lysines of target proteins. Here we demonstrate that attachment of SUMO to the N-terminus of under-expressed proteins dramatically enhances their expression in E. coli. SUMO protease 1 was able to cleave a variety of SUMO fusions robustly and with impeccable specificity. Purified recombinant SUMO-GFPs were efficiently cleaved when any amino acid, except proline, was in the+1 position of the cleavage site. The enzyme was active over a broad range of buffer and temperature conditions. Purification of certain recombinant proteins is accomplished by production of Ub-fusions from which Ub can be subsequently removed by de-ubiquitinating enzymes (DUBs). However, DUBs are unstable enzymes that are difficult to produce and inexpensive DUBs are not available commercially. Our findings demonstrate that SUMO protease 1/SUMO-fusion system may be preferable to DUB/Ub-fusion. Enhanced expression and solubility of proteins fused to SUMO combined with broad specificity and highly efficient cleavage properties of the SUMO protease 1 indicates that SUMO-fusion technology will become a useful tool in purification of proteins and peptides.     

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.     

Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.     

Transcriptional Profiling of Gene Expression Changes in a PACE-Transfected CHO DUKX Cell Line Secreting High Levels of rhBMP-2

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry for the production of recombinant human proteins including complex polypeptides such as recombinant human bone morphogenic protein 2 (rhBMP-2). Large-scale manufacture of rhBMP-2 has associated production difficulties resulting from incomplete processing of the recombinant human protein due to insufficient endogenous levels of the paired basic amino acid cleaving enzyme (PACE) in CHO. In order to resolve this issue, CHO DUKX cells expressing rhBMP-2 were transfected with the soluble version of human PACE (PACEsol) resulting in improved amino-terminal homogeneity and a fourfold increase in rhBMP-2 productivity. In this article, we present a microarray expression profile analysis comparing the parental lineage to the higher producing subclone co-expressing PACEsol using a proprietary CHO-specific microarray. Using this technology we observed 1,076 significantly different genes in the high-productivity cells co-expressing PACEsol. Following further analysis of the differentially expressed genes, the Unfolded Protein Response (UPR) component of the endoplasmic reticulum stress response pathway was identified as a key candidate for effecting increased productivity in this cell system. Several additional ER- and Golgi-localised proteins were identified which may also contribute to this effect. The results presented here support the use of large-scale microarray expression profiling as a viable and valuable route towards understanding the behaviour of bioprocess cultures in vitro.     

真核表达MERS-CoV刺突蛋白亚单位的信号肽序列优化研究

中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的刺突蛋白(Spike,S)亚单位1(S1)是引起宿主免疫反应和产生中和抗体的主要靶抗原,也是疫苗研发和病原检测的重要靶标,选用适宜的真核表达系统高效表达S1蛋白是进行相关研究的基础。为确定MERS-CoV S1在哺乳动物细胞中高效分泌性表达的信号肽序列,构建了含高斯荧光素酶(Gaussia luciferase,GLuc)、人组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)及小鼠免疫球蛋白G的2a亚型(Mouse immunoglobular G subtype 2a,MIgG2a)7个信号肽(原始序列和改造序列)序列的MERS-CoV S1表达质粒,瞬时转染细胞后,通过Western Blot检测并比较细胞培养上清和裂解液中S1的表达水平及分泌表达效率(条带密度灰度扫描比),并对哺乳动物细胞表达的S1蛋白的纯度与抗原特性进行了分析。结果表明7种信号肽在293T、BHK21和ExpiCHO-S^TM三种细胞系统中介导MERS-CoV S1的高效分泌表达的效率各有不同,其中tPA-1信号肽介导S1抗原在ExpiCHO-S^TM中具有较高的分泌表达效率与产量,纯化的S1蛋白保持了较好的抗原性。本研究为进一步研发基于MERS-CoV S1的亚单位疫苗及免疫学检测试剂奠定了基础。     

SARS亚基疫苗-突刺蛋白受体结合区RBD在烟草叶绿体中的高效表达

叶绿体表达系统为植物源重组药用蛋白和亚基疫苗的生产提供了一个有效的途径。为验证SARS亚基疫苗在叶绿体中表达的可行性,以及为植物源SARS亚基疫苗的生产提供一套高效、低成本的技术平台,本研究将人工优化合成的SARS-CoV突刺蛋白(S蛋白)受体结合区序列RBD与载体分子CTB融合基因导入烟草叶绿体基因组中。PCR和Southern杂交分析表明,外源融合基因已整合到烟草叶绿体基因组中,并获得同质化。Western杂交分析表明,重组融合蛋白CTB-RBD在叶绿体转基因烟草中获得表达,且主要以可溶性单体形式存在。ELISA分析表明,在不同生长阶段、不同生长部位和不同时间点烟草叶片中,重组融合蛋白CTB-RBD的表达水平呈现明显的变化。重组蛋白在成熟叶片中的表达水平最高可以达到10.2%TSP。本研究通过SARS亚基疫苗RBD在烟草叶绿体中的高效表达,有望为植物源SARS亚基疫苗的生产以及SARS血清抗体的检测提供一个有效的技术平台。