Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.     

重组PLZF蛋白锌指结构域的表达、提纯和活性分析

PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成。鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯。为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点。构建好的表达质粒转化到BL21 (DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达。用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上。对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础。     

High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure

Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely. To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far. We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli. Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient. With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins. This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus. Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction. This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis. To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form. The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin. In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs.     

Development of an improved preparation and an enzyme-linked immunosorbent assay for anti-neuroexcitation peptide (ANEP)

Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His(6) encoding recombinant ANEP with a His(6)-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.     

Mouse methionine sulfoxide reductase B: effect of selenocysteine incorporation on its activity and expression of the seleno-containing enzyme in bacterial and mammalian cells

The mammalian methionine sulfoxide reductase B (MsrB) has been found to be a selenoprotein which can reduce R form of both free and protein-incorporated methionine sulfoxide to methionine. Together with MsrA, which reduces specifically the S form of methionine sulfoxide, the living cell can repair methionine-damaged proteins and salvage free methionine under oxidative stress conditions. Here, we report about the pivotal role of the selenocysteine residue in the protein putative active site by site-directed mutagenesis directed to the selenocysteine codon. Using the Escherichia coli SECIS (selenocysteine insertion sequence) element, needed for the recognition of the UGA codon as a selenocysteine codon in E. coli, we expressed the seleno-MsrB as a recombinant selenoprotein in E. coli. The recombinant seleno-MsrB has been shown to be much more active than the cysteine mutant, whereas the mutations to alanine and serine rendered the protein inactive. Although the yields of expression of the full-length N-terminus and C-terminus His-tagged seleno-MsrB were only 3% (of the total MsrB expressed), the C-terminus His-tagged protein enabled us to get a pure preparation of the seleno-MsrB. Using both recombinant selenoproteins, the N-terminus His-tagged and the C-terminus His-tagged proteins, we were able to determine the specific activities of the recombinant seleno-MsrB, which were found to be much higher than the cysteine mutant homologue. This finding confirmed our suggestion that the selenocysteine is essential for maintaining high reducing activity of MsrB. In addition, using radioactive selenium we were able to determine the in vivo presence of MsrB as a selenoprotein in mammalian cell cultures.     

Expression of anti-neuroexcitation peptide (ANEP) of scorpion Buthus martensii Karsch in Escherichia coli

According to the cDNA sequence of anti-neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti-neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by a PCR method, then was cloned into expression plasmid pET28a, fused with His tag at its 3' end. When expressed in E. coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins, while most recombinant ANEP products existed in the form of insoluble inclusion bodies. Coexpression of molecular chaperones or protein disulfide isomerase could not improve its solubility. The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and Superdex 30 chromatography. In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti-neuroexcitatory activity.     

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.     

Expression of His-tagged Shigella IpaC in Arabidopsis

Although the expression of histidine (His)-tagged proteins in bacteria is routine, few His-tagged proteins have been expressed in plants, and no His-tagged proteins from bacterial pathogens have been expressed in plants, to our knowledge. Here, we demonstrate expression of the Shigella flexneri invasion plasmid antigen, IpaC, in Arabidopsis thaliana. S. flexneri is the causitive trigger for bacillary dysentery, and IpaC is essential for bacterial entry into epithelial cells. IpaC, attached to a 5' leader containing six tandem His codons, was cloned into a pBI121 vector. This clone was introduced into Agrobacterium tumefaciens and Arabidopsis plants were then transformed. T1 and T2 plant generations were obtained. Total plant proteins were extracted from T2 leaves; the Bradford assay was used to determine protein concentrations. A nickel-coated ELISA plate method, using both anti-His and anti-IpaC 1 degrees antibodies, was used to detect and quantify IpaC in transgenic Arabidopsis plants. Between 1.9 and 2.3 microg IpaC/mg total plant protein was obtained; this equals 0.2% of total protein, an amount comparable to other recombinant protein estimates in plants. Expressing His-tagged proteins from bacterial pathogens, in plants, is important because plant material could ultimately be fed or applied intranasally to animals that are "at risk" for infection by such bacterial pathogens, thus causing them to raise antibodies against the pathogens--functioning as a vaccine.     

Development of an improved preparation and an enzyme-linked immunosorbent assay for anti-neuroexcitation peptide (ANEP)

Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His₆ encoding recombinant ANEP with a His₆-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.     

Amino acid sequence of human respiratory syncytial virus nucleocapsid protein.

Amino acid sequence of the human respiratory syncytial (RS) virus nucleocapsid (NC) protein, deduced from the DNA sequence of a recombinant plasmid, is presented. The cDNA plasmid (pRSB11) has 1412 bp of RS viral NC sequence and lacks six nucleotides of the 5' end of mRNA. There is a single long open reading frame encoding 467 amino acids. This 51540 dal protein is rich in basic amino acids and has no homologies with other known viral capsid proteins.     

霍乱毒素B亚基融合蛋白表达系统的构建

霍乱毒素Ｂ亚基（ＣＴＢ）是良好的免疫佐剂和载体蛋白。本研究通过定点突变,在ＣＴＢ基因（ｃｔｘＢ）３′端终止密码前引入了限制性内切酶ＥｃｏＲＩ,构建了质粒ｐＭＣ０５。ｐＭＣ０５中ＣＴＢ与下游ｌａｃＺ′基因阅读框架相同,转化大肠杆菌后能够表达ＣＴＢ与β－半乳糖苷酶α肽的融合蛋白;所表达的融合蛋白能与ＧＭ１结合,说明融合蛋白保持ＣＴＢ的基本高级结构和生物学活性;融合蛋白能与抗－ＣＴＢ抗体结合,说明融合蛋白具有ＣＴＢ的抗原性。以上结果表明：通过将外源抗原决定簇基因融合至ｃｔｘＢ的３′端,在大肠杆菌中表达融合蛋白,构建基因工程肽苗是可行的。还探索了转录终止序列对融合基因蛋白表达水平的影响,构建了高效表达融合蛋白的载体－宿主系统。     

抗菌肽Cecropin B-人溶菌酶融合蛋白表达载体的构建

目的是构建抗菌肽B(Cecropin B)和人溶菌酶(hLyso)的融合蛋白表达载体。从pUC118～hLyso上卸下人溶菌酶基因后,通过重叠区扩增法人工合成抗菌肽B基因,并将其融合到人溶菌酶基因的5’端。将抗菌肽B基因和人溶菌酶基因按正确的阅读框架定向克隆至大肠杆菌高效表达载体pET32a,终止子位于人溶菌酶基因的3’端。PCR鉴定及序列分析表明,所转化的BL21(DE3)菌落中含有插入Cecropin B-hLyso基因的重组质粒pET32a-CB-hLyso。     

Sequence analysis of the respiratory syncytial virus phosphoprotein gene.

A recombinant cDNA plasmid (pRSA3) containing an almost full-length copy of the mRNA encoding respiratory syncytial virus phosphoprotein was identified in a cDNA library prepared with mRNA from respiratory syncytial virus-infected cells. The cDNA insert was sequenced, and a protein of 27,150 daltons was deduced from the DNA sequence. The protein is relatively acidic, containing two clusters of acidic amino acids, one in the middle of the molecule and the other at the C-terminus. It is devoid of both cysteine and tryptophan. There was no other potential reading frame within the phosphoprotein gene of respiratory syncytial virus. This situation is unlike that with Sendai virus, a paramyxovirus, which has a nonstructural C protein encoded by a second overlapping reading frame near the 5' end of the mRNA for phosphoprotein.     

pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants

Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5'-untranslated region (UTR) and the 3'-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV- HT ) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved [ Sainsbury, F. and Lomonossoff, G.P. (2008) Plant Physiol . 148 , 1212–1218]. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of non-essential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the production of milligram quantities of recombinant proteins from plants with standard plant research techniques at a bench-top scale.     

Intein介导的重组昆虫毒素BmK IT在大肠杆菌中的可溶性表达、纯化及活性分析

为获得重组蝎昆虫毒素BmKIT,通过PCR方法在BmKIT基因的3′端融合了编码6个组氨酸残基的核苷酸序列,将其插入原核表达载体pTWIN1的内含肽Ssp DnaB Intein基因下游的多克隆位点(MCS)。将获得的表达质粒转化大肠杆菌BL21(DE3)中,用IPTG诱导融合蛋白表达。用Ni-NTA亲和层析柱从菌体裂解液中纯化了CBD-Intein-BmK IThis6融合蛋白,并在柱上诱导Intein自剪切,成功去除融合子CBD-Intein。通过Superdex75凝胶过滤层析获得了纯度达95%以上的BmK IThis6蛋白,该蛋白不仅具有正确的二级结构而且有生物活性。