Expression of the Canavalia brasiliensis lectin (ConBr) in tobacco plants

Tobacco plants were transformed with gene constructs encoding prepro-ConBr (Canavalia brasiliensis lectin). Transgenic plants confirmed by PCR expressed the recombinant protein as revealed by Western blot. However, the apparent molecular mass of the recombinant polypeptide (ca. 34 kDa) was higher than the native lectin (about 30 kDa), showing that further proteolytic processing of pro-ConBr was not detected.     

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.     

ConBr, a Lectin from Canavalia brasiliensis Seeds, Protects Against Quinolinic Acid-Induced Seizures in Mice

Lectins are proteins capable of reversible binding to carbohydrates or glycoconjugates. In the central nervous system of mammals, lectins with affinity for mannose/glucose or galactose can modulate cellular communication. ConBr, a lectin isolated from the seeds of Canavalia brasiliensis, previously showed antidepressant effect in the forced swimming test in mice, with involvement of the monoaminergic system. In this study, we investigated the neuroprotective effects of ConBr against quinolinic acid (QA), a well-known NMDA agonist that produces severe neurotoxicity when administered in vivo. ConBr (10 μg/site) administered via intracerebroventricular (i.c.v.) showed a neuroprotective activity against seizures induced by QA (36.8 nmol/site; i.c.v.) when administered 15 min prior to QA, with a percentage of protection around 50%. ConBr was also able to significantly decrease the severity of the seizures but without changes in the latency of the first convulsion or the duration of the seizures. This effect was dependent on the structural integrity of the ConBr protein and its binding capacity to oligosaccharides residues. ConA, a lectin with high similarity to ConBr, did not reverse the QA-induced seizures. Moreover, ConBr was able to protect against hippocampal cell death caused by QA, which was measured by propidium iodide incorporation. QA caused activation of JNK2 and improved the phosphorylation of Ser831 and 845 on the AMPA receptor GluR1 subunit, and both of these effects were counteracted by ConBr. Our data suggest that the lectin ConBr may exert a modulatory action on NMDA receptors, which inhibits its activity in response to QA.     

Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris

The Nicotiana tabacum lectin, also called Nictaba, is a nucleocytoplasmic plant lectin expressed in tobacco leaves after exposure to jasmonates. Purification of the lectin from raw material is a time-consuming process, demanding large amounts of induced plant material. In addition, the lectin yield is low and purified lectin fractions are always contaminated with low molecular weight compounds such as phenols. In a way to improve and facilitate the purification of the tobacco lectin, we cloned the Nictaba gene in a vector optimized for protein expression in the methylotrophic yeast Pichia pastoris. In this report, we present data of the expression profile of recombinant Nictaba in the P. pastoris culture medium and in P. pastoris cells together with the purification strategy using ion exchange chromatography and affinity chromatography on a column with immobilized ovomucoid. Pichia transformants were estimated to express approximately 6mg of recombinant lectin per liter medium after a 72h culture. SDS-PAGE and Western blot analysis revealed that the recombinant lectin expressed in Pichia exists in two molecular forms. Edman degradation and mass spectrometry analysis confirmed the presence of at least two forms of recombinant lectin with molecular weights of 19,060 and 20,100Da, corresponding to lectin polypeptides similar to the fully processed Nictaba which is N-terminally blocked, and Nictaba extended at the N-terminus with the amino acids residues EAEAYVEFT due to incomplete processing of the alpha-factor mating sequence. Further characterisation of the recombinant lectin revealed agglutination and carbohydrate-binding properties similar to the native tobacco lectin.     

Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris

Rice (Oryza sativa) expresses different putative carbohydrate-binding proteins belonging to the class of lectins containing an Euonymus lectin (EUL)-related domain, one of them being OrysaEULS2. The OrysaEULS2 sequence consists of a 56 amino acid N-terminal domain followed by the EUL sequence. In this paper the original sequence of the EUL domain of OrysaEULS2 and some mutant forms have been expressed in Pichia pastoris. Subsequently, the recombinant proteins were purified and their carbohydrate binding properties determined. Analysis of the original protein on the glycan array revealed interaction with mannose containing structures and to a lesser extent with glycans containing lactosamine related structures. It was shown that mutation of tryptophan residue 134 into leucine resulted in an almost complete loss of carbohydrate binding activity of OrysaEULS2. Our results show that the EUL domain in OrysaEULS2 interacts with glycan structures, and hence can be considered as a lectin. However, the binding of the protein with the array is much weaker than that of other EUL-related lectins. Furthermore, our results indicate that gene divergence within the family of EUL-related lectins lead to changes in carbohydrate binding specificity.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

肿瘤坏死因子相关凋亡诱导配体(sTRAIL)在毕赤酵母中的高效表达

从人胎盘总RNA中通过RTPCR方法获得sTRAIL基因的cDNA,并通过构建高拷贝表达载体在毕赤酵母中获得了高效表达,表达量可达40.1mg/L。并对表达产物进行了分离纯化和生物学活性分析,获得了纯度大于90%的纯品,该样品能明显表现出诱导L929肿瘤细胞凋亡的作用,半数致死量为0.18μg/mL,与文献报道的大致相同。     

Expression and purification of a biologically active Phytophthora parasitica cellulose binding elicitor lectin in Pichia pastoris

The Phytophthora parasitica cellulose-binding elicitor lectin, (CBEL), is a cell wall-localized protein playing a key role in cell wall organization and adhesion of the mycelium to cellulosic substrates. CBEL is a potent elicitor of plant immune responses and this activity is linked to its ability to bind plant cell wall components. In order to scale up the production of active CBEL, we reported here the cloning and expression of a His-tagged version of CBEL in the yeast Pichia pastoris. Selection of a high-producing P. pastoris clone and optimization of the purification procedure allowed a yield of about 2mg of pure protein per liter of culture filtrate. The identity of the recombinant protein was confirmed by western-blot analysis, N-terminal protein sequencing, and by peptide mass fingerprinting. The cellulose-binding affinity and the lectin activity of the recombinant protein were identical to the native CBEL. Its elicitor activity, tested on Arabidopsis thaliana leaves, was similar to the native CBEL protein as it displays a similar biological activity on plant immune responses inducing defense gene expression and localized necroses of the infiltrated leaf tissues. The present work suggests that P. pastoris can be a suitable host for the production of compounds active on plants or for the development of new agricultural products able to stimulate plant immunity.     

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Key words proinsulin; C-peptide; Pichia pastoris     

Expression of frutalin, an alpha-D-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

Frutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.     

一种新型巴斯德毕赤酵母表达载体的构建及甘露聚糖酶的表达

目的:构建以带自身启动子的蔗糖转化酶基因(suc2)为选择标记的载体,用于外源基因在巴斯德毕赤酵母中的正确分泌表达。方法:根据已发表的蔗糖转化酶基因序列设计并合成1对引物,应用PCR技术,以啤酒酵母INVSC1总DNA为模板,扩增出包含自身启动子和终止区序列的suc2基因。将该基因与毕赤酵母表达载体pPIC9K连接,构建了以suc2为选择标记的表达载体pPIC12K。将甘露聚糖酶基因man克隆入载体pPIC12K,用PEG/LiCl法转化毕赤酵母GS115菌株。以蔗糖为惟一碳源筛选转化子,利用底物平板检测筛选到的转化子中man基因的表达,并对重组表达菌株进行连续传代实验。结果:部分转化子周围产生明显的水解圈,证明甘露聚糖酶已经得到分泌表达;对重组表达菌株的连续传代实验证实了该表达载体具有良好的遗传稳定性。结论:以带自身启动子的suc2基因为选择标记的表达载体构建成功,并且这个新型表达载体能够对外源基因进行稳定有效的分泌表达。     

Expression of a cold-adapted fish trypsin in Pichia pastoris

Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success and are contained mainly in patent applications, which disregard the difficulties associated with the developments. Although the yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin, it has shown problems when expressing cold-adapted fish trypsins (CAFTs). CAFTs are considered of immense value for their comparative advantage over other trypsins in a number of food-processing and biotechnological applications. Thus, to investigate potential obstacles related to the production of CAFTs in P. pastoris, the cunner fish trypsin (CFT) was cloned in different Pichia expression vectors. The vectors were constructed targeting both internal and secreted expression and keeping the CFT native signal peptide. Western-blotting analysis confirmed the expression with evident differences for each construct, observing a major effect of the leader peptide sequence on the expression patterns. Immobilized nickel affinity chromatography yielded a partially purified recombinant CFT, which exhibited trypsin-specific activity after activation with bovine enterokinase.     

Expression of lignocellulolytic enzymes in Pichia pastoris

ABSTRACT: BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.     

人β-分泌酶 (BACE1) 在毕赤酵母中分泌表达及纯化

为了获得活性良好的重组人β-分泌酶 (β-secreatase, BACE1),用于研究其与抑制剂的作用模式,构建了携带β-分泌酶proBACE1和BACE1编码序列的重组表达质粒pPIC9K-MetBACE22和pPIC9K-MetBACE46,通过电击法转入毕赤酵母GS115中,分别得到重组子9k-B22和9k-B46。重组菌株在诱导表达培养基中诱导外源基因表达,结果显示9k-B22的上清活性明显高于9k-B46的上清活性。9k-B22表达上清浓缩后经HisTrap亲和柱纯化得到的蛋白具有良好的BACE1活性, SDS-PAGE/高碘酸-希夫试剂染色发现其为糖蛋白,并且其糖基侧链可以被Endo Hf完全切除,得到50 kDa左右的两条蛋白带。肽质量指纹图谱鉴定发现,这两个蛋白分别与proBACE1和BACE1匹配。活性检测发现糖基化BACE1和去糖基化BACE1的活性均低于HEK-293细胞表达的商品BACE1,这说明糖基化及其类型对BACE1的活性非常重要。然而已知的BACE1抑制剂对三者的抑制率无显著差异,这说明糖基化并不影响与抑制剂的相互作用。经过一系列培养条件优化BACE1纯化产量提高到1 mg/L,这为发现并优化BACE1新型抑制剂的相关研究奠定了物质基础。     

重组瑞替普酶在毕赤酵母中的表达

【目的】瑞替普酶(重组组织纤溶酶原激活物,rt PA)被认为是第三代安全有效的溶栓剂,以p PIC9K为载体,以3种不同表型的毕赤酵母(Pichia pastoris)为宿主,探索适合rt PA分泌型表达的最佳体系。【方法】以质粒p ET28a-rt PA为模板,设计特异性引物,PCR扩增目的基因rt PA,插入分泌型表达载体p PIC9K中,获得重组表达质粒p PIC9K-rt PA。重组质粒经限制性内切酶Sal I线性化后,电击转化至3种不同表型的P.pastoris(GS115、SMD1168、KM71)中进行组成型表达;重组表达体系进行甲醇诱导表达,对产物进行Western blot鉴定,并采用纤维蛋白平板溶圈法测定其活性。【结果】重组蛋白分子量约为43 k D;rt PA-GS115和rt PA-KM71均在39 k D处有特异性条带,且前者在32 k D处有轻微降解条带,而后者并无此现象;rt PA-SMD1168无降解现象,且rt PA-SMD1168比活性较rt PA-GS115高27%;rt PA-KM71表达量和活性均为最低。【结论】从重组蛋白生物活性出发,P.pastoris SD1168可作为rt PA的最佳表达体系,在控制宿主蛋白酶活性、减少产物降解的前提下,P.pastoris GS115也是rt PA表达的优选体系。     

Expression and characterization of a variant of TACI (CRD2-shortTACIFc) in Pichia pastoris

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. The extracellular domain of a typical TNFR contains multiple copies of CRD, which bind in the monomermonomer interfaces of a trimeric ligand. TACI binds to two ligands, APRIL and BAFF, with high affinity and contains two CRD in its extracellular regions, while BCMA and BR3, contain a single or partial CRD for binding the two ligands. However, TACI can be classified as a single CRD receptor because the amino-terminal CRD1 doesn't contribute to ligand binding. To obtain a new variant of TACI possessing higher affinities for binding, we fused a repeat sequence of CRD2 to the N-terminus of the short form of TACI. The new APRIL antagonist peptide, CRD2-shortTACI-Fc, was designed based on the modeling 3-D complex structure of TACI and APRIL. As expected, the purified recombinant CRD2-shortTACI-Fc fusion protein could bind to APRIL in vitro and demonstrated dose-dependent inhibition of APRIL-induced proliferative activity in Raji cells. We found that CRD2-shortTACI-Fc, has a higher affinity for binding to ligands than short-TACI-Fc, which contains a single CRD2.     

一个推测的拟南芥漆酶基因在巴斯德毕赤酵母中的表达

At5g01040基因是一个推测的拟南芥漆酶基因。根据其编码序列设计引物,利用RT—PCR方法扩增出1755bp的片段。测序结果表明,该片段存在3个突变位点,其中一个突变位点改变了所编码的氨基酸。将该片段克隆到表达载体pPICZaB上,电击转化毕赤酵母。经筛选获得80个候选重组菌株,其中18个菌株的培养液中存在漆酶活性,说明所克隆的基因在毕赤酵母中实现了分泌表达,证实了At5g01040编码的蛋白具有漆酶活性。     

TIMP-2在毕赤酵母中的克隆与表达

为了克隆人基质金属蛋白酶组织抑制剂-2（TIMP-2）基因,并在Pichia pastoris中表达,根据GenBank上的TIMP-2的氨基酸序列和毕赤酵母偏爱密码子,通过化学合成和PCR相结合的方法获得了人的TIMP-2基因全长序列,构建了pPIC9-T2表达载体,电击转化到毕赤酵母,通过表型筛选和诱导表达得到蛋白表达工程菌,并对表达产物进行了分离纯化和生物学活性分析。     

外源基因在毕赤酵母中表达的优化

巴斯德毕赤酵母是近年来成功的外源基因表达系统,已表达出众多外源蛋白。它既能像原核生物一样快速生长高密度发酵,又能进行真核翻译后修饰,并且蛋白分泌量大,因此应用越来越广泛。如果对它的表达载体,转化诱导条件和目的基因内部结构,发酵条件等方面进行优化,能够进一步发挥它的优势,更好地表达需要的外源蛋白。本文就毕赤酵母表达系统表达优化进行总结综述。