Expression and Purification of Neurotoxin-Associated Protein HA-33/A from Clostridium botulinum and Evaluation of Its Antigenicity

Background: Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. Methods: Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. Results: The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. Conclusion: The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice. Key Words: Botulinum neurotoxin, Expression, Purification     

Population dynamics of Xanthomonas campestris pv. vitians on different plant species and management of bacterial leaf spot of lettuce under greenhouse conditions

The population dynamics of Xanthomonas campestris pv. vitians (Xcv) was studied both externally and internally in lettuce, tomato and pepper plants. In addition, the application of bactericides during transplant production period was carried out for the management of bacterial leaf spot of lettuce under greenhouse conditions. Epiphytic populations of Xcv were recovered on leaves of lettuce plants (10⁵ CFU/g) 5 weeks after sprayed than the other plant species when inoculated with 10⁸ CFU/ml of Xcv. When plants of each crop species infiltrated with the bacterium at 10⁵ CFU/ml, the highest populations were developed in lettuce (10⁸ CFU/cm²) followed by pepper with 10⁶ CFU/cm² and tomato with 10⁵ CFU/cm² 10-days after infiltration. Application of a mixture of Maneb and Kocide or Kocide alone as a foliar spray on lettuce significantly reduced the incidence and disease severity of bacterial leaf spot by 29 and 27% respectively. Spread of the bacterium and development of the disease may partly be managed by avoiding intercropping of these plants commonly grown in close proximity to lettuce. For the management of leaf-associated populations of Xcv in lettuce, use of a mixture of Maneb and Kocide is advocated to minimize the effect of attacks.     

Efficient Process Development of Recombinant Human Granulocyte Colony-Stimulating Factor (rh-GCSF) Production in Escherichia coli

Background:

The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients.

Methods:

An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h^-1 at the beginning of feeding to 0.4 h^-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography.

Results:

Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l^-1 and 32.1 g.l^-1. Also, the final specific yield (Y_P/X) and total productivity of rh-GCSF were obtained 254 mg.g^-1 DCW and 1.83 g.l^-1.h^-1, respectively. According to the available data, this is one of the highest Y_P/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen® and PD-Grastim showed that all proteins have a similar secondary structure.

Conclusion:

Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity. Key Words: Escherichia coli, Granulocyte colony-stimulating factor (GCSF), Process development     

Cloning,Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium,Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS-PAGE. Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results:Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS- PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion:In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity. Key Words: Escherichia coli, Gene expression, Lactobacillus, Lipase, Phylogeny     

Cloning and expression analysis of kenaf (Hibiscus cannabinus L.) major lignin and cellulose biosynthesis gene sequences and polymer quantification during plant development

In order to apply state-of-the-art molecular breeding techniques in fibre crop it is necessary to have a good knowledge of major polymer biosynthesis gene sequences and their expression pattern. Polymerase chain reaction was employed to isolate sequences of the major genes for lignin and cellulose biosynthesis in a kenaf cultivar. CeSA, 4cl, c4h, cad, and ccr gene primers were designed according to their conservative regions; partial sequences of lignin and cellulose biosynthesis genes were obtained. One actin II gene sequence was also isolated from the kenaf genome as a housekeeping gene to be employed in qPCR analysis. Expression levels of genes c4h, cad and CeSA in bark and core from plants harvested at three different growth stages were evaluated. Using qPCR analyses it was found that the expression levels of the two biosynthesis lignin genes in bark tissues increased during plant growth, while a negative trend was recorded in core tissues. In both bark and core, the quantity of lignin was positively correlated to plant growth while cellulose content decreased.     

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully. Key Words: Pteridine reductase 1 (PTR1), Leishmania, Gene expression     

Development of Bt transgenic potatoes for effective control of potato tuber moth by using cry1Ab gene regulated by GBSS promoter

Transgenic of Indian potato cultivar Kufri Badshah expressing synthetic, modified cry1Ab gene were developed against potato tuber moth (Phthorimaea opercullela Z.) a destructive pest. The cry1Ab gene was in spatial and temporal expression under the control of tuber-specific GBSSi promoter. The transformation vector pBinCG1 was developed harbouring transgene expression cassette comprising cry1Ab gene under the control of potato GBSSi promoter, castor bean catalase intron (5′UTR) and OCS termination signals. Selected kanamycin-resistant putative transformed potato lines were evaluated by positive PCR amplification of 713 bp, 1206 bp and 700 bp with npt II, GBSSi promoter and cry1Ab gene specific primers, respectively. Gene integration in transformed potato plants was elucidated through Southern hybridization and in planta transgene expression analysis. Transgene expression was highest in stolons, followed by tubers matured leaves and young leaves. Insect bioassay of stored transgenic tubers resulted in significant retardation and mortality in neonate tuber moth larvae. RT-PCR and northern hybridization confirmed stable expression in stored tubers. These transgenic lines; thus can represent an effective resistance management strategy which can significantly reduce the selection pressure on target and non-target insect pests to a threshold level.     

Marker gene excision in transgenic Brassica napus via Agrobacterium-mediated Cre/lox transient expression system

Oilseed rape (Brassica napus L.) is one of the most important oil crops in the world. However, study on marker-free transgene of B. napus for bio-safety purpose is limited in this allotetraploid crop. In order to advance marker gene excision research, a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B. napus. The system consists of 2 sets of independent vectors including pC35Spro::T7RP carrying T7 RNA polymerase and pCT7pro::Cre carrying T7 promoter respectively. After hybridization of 2 according types of transgenic B. napus, marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression. Totally 52 and 46 positive T₀ transgenic lines of these 2 vectors were obtained after identification by PCR and test trip. T₁ plants from 3 T₀ positive pC35Spro::T7RP lines and 2 T₀ positive pC35Spro::T7RP lines were identified as single copy according to segregation ratio and were chosen for crossing. However, expression of CP4 EPSPS (glyphosate resistance gene) and OXY (bromoxynil resistance gene) were not found in F₁ progeny, which proved that the excision was not complete. The possible reasons for our limited success were investigated and detailed analyses were performed. Although this system is not applicable for generating transgenic B. napus free from selectable marker gene, it provided valuable experience and clue for further improvement of this technique. Many other advantages and further improvement will be progressed in future work.     

Evaluation of the short term effect of nursery treatments with phosphite-based products,acibenzolar-S-methyl,pelleted Brassica carinata and biocontrol agents,against lettuce and cultivated rocket fusarium wilt under artificial inoculation and greenhouse conditions

Experimental trials have been carried out in order to evaluate the efficacy of preventative treatments based on plant defense activator products, biocontrol agents, a microbial complex with arbuscular mycorrhizal fungi, and Brassica carinata pellets against Fusarium oxysporum f. sp. lactucae race 1 on lettuce and Fusarium oxysporum f. sp. raphani on cultivated rocket under greenhouse conditions. These products were compared with fungicides known for their ability to induce host resistance (phosethyl-Al and acibenzolar-S-methyl), and with azoxystrobin. Three and four applications of the tested products were carried out on lettuce and rocket seedlings grown in nursery conditions. Treated and untreated plants were transplanted into soil infested with Fusarium wilt agents to obtain an average disease severity (DS) of 65.6–69.2 and of 56.9–62.1 on the untreated lettuce and rocket plants, respectively. The best Fusarium wilt biocontrol was obtained after four applications of Bacillus subtilis Qst713 and with the Glomas microbial complex (42 and 46.7%, efficacy, respectively). B. carinata pellets provided a consistent control when applied 14 days before the rocket and lettuce were transplanted into the infested soil. Acibenzolar-S-methyl, applied at 0.025 g/Liter, showed a DS reduction in F. oxysporum f. sp. lactucae from 36 to 61% and of F. oxysporum f. sp. raphani from 54 to 73%, thus showing statistically similar results to those of azoxystrobin, which was used as a reference (DS reduction from 59 to 65%). Although the Fusarium wilt control provided by such products was not complete in the present experimental conditions, these products can be considered interesting components for an integrated pest management of the Fusarium wilt of leafy vegetables, starting from nursey applications. Moreover, the tested BCAs could become potentially useful, especially for plant monocultures. This study has been produced new information on the effects of potassium phosphite, applied at the nursery level, on reducing lettuce and rocket fusarium wilt. An average efficacy of 69.5% was observed for lettuce, while an average efficacy of 65.2% was observed for cultivated rocket. The good fungicidal activity of the phosphite-based product, coupled with the positive effect on plant biomass, is of special interest.     

Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number {"type":"entrez-nucleotide","attrs":{"text":"AY729671","term_id":"56718401","term_text":"AY729671"}}AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.     

Phosphomannose isomerase,pmi, as a selectable marker gene for durum wheat transformation

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional methods. The development of transgenic plants requires the use of selectable marker genes, as the efficiency of plant transformation is less than optimal for many important species, especially for monocots such as durum wheat (Triticum turgidum var. durum). Many concerns have been expressed about the persistence of currently used marker genes in plants used for field cultivation. To sustain further progress in this area, alternative efficient selection methods are desirable. A recent development is the use of selective genes that give transformed cells a metabolic advantage (positive selection) compared to untransformed cells, which are slowly starved with a concomitant reduction in growth and viability. This selection strategy is in contrast to traditional negative selections during which the transgenic cells are able to survive on a selective medium whereas the non-transgenic cells are actively killed by the selective agent. We compared the ‘selection efficiency’ of a commonly used negative selection method that employs the Streptomyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos, to a positive selection employing the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable gene and mannose as the selective agent. Calli derived from immature embryos of the durum wheat cultivar Svevo were bombarded separately with bar and pmi genes using a biolistic system. The integration and expression of the two genes in the T₀ generation were confirmed by PCR analysis with specific primers for each gene and the chlorophenol red assay, respectively. The selection efficiency, calculated as the number of expressing plants divided by the number of total regenerants, was higher when pmi was used as the selectable marker gene (90.1%) than when bar was used (26.4%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide résistance genes.     

Kenaf (Hibiscus cannabinus L.) based substrates for the production of compact plants

Growth media based on whole-stem kenaf (Hibiscus cannabinus L.) and sand have been used to produce compact lettuce (Lactuca sativa L.) and pepper (Capsicum annuum L.) plants. Seeds were sown directly in kenaf-containing substrates and growth was recorded for up to 100 days after sowing. The presence of whole-stem kenaf (core and bark), even at a ratio of 10:90 (kenaf:sand), inhibited plant growth expressed as plant height, leaf number, and plant fresh and dry weight. When plants were subsequently transplanted to a kenaf-free substrate, growth continued at a similar rate to that of the control (sown and grown in peat and sand). The inhibitory effect of kenaf is located both in the core and bark, but is decreased by soaking the kenaf in NH₄NO₃ prior to use. A possible role for whole-stem kenaf (core and bark) in the production of compact plants is proposed.     

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

Background:

Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.

Results:

The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.

Conclusion:

Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)     

Utilization of Site-Specific Recombination in Biopharmaceutical Production

Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons.     

重组人血清白蛋白在紫花苜蓿中的转基因表达

为获得高产重组人血清白蛋白的转基因紫花苜蓿"游客"(Medicago sativa L. cv.‘Eureka’)株系用于规模化生产rHSA,构建了rHSA植物表达载体。以紫花苜蓿子叶愈伤组织为受体,通过农杆菌介导法进行遗传转化,筛选出潮霉素抗性植株,提取抗性再生紫花苜蓿植株基因组DNA做PCR鉴定,结果表明重组人血清白蛋白基因片段已整合到紫花苜蓿基因组中;提取转基因植株总蛋白,Western blotting检测结果表明rHSA在转基因植株中成功表达。此结果表明转基因紫花苜蓿可稳定表达rHSA。      

PCR-based markers for identification of HMW-GS at Glu-B1x loci in common wheat

The bread wheat elasticity, which is very important for bread-making quality, is largely determined by the composition of high-molecular-weight glutenin subunits (HMW-GS). The HMW-GS encoded by Glu-B1 loci are highly polymorphic and the combinations 17+18 and 14+15 are good for bread making. Thus it is very important to identify the alleles at Glu-B1 loci for wheat quality improvement. In this study, the five common HMW-GS types encoded by Glu-B1x locus carried by 18 Chinese bread wheat cultivars (or lines) were analyzed by SDS-PAGE. Two pairs of PCR primers which could distinguish the Glu-Blx alleles of the five common HMW-GS types were designed based on the Glu-B1x gene sequences (Reddy and Appels, 1993; Genbank accession: X13927; Genbank accession:AY367771). 22 recombinant inbred lines (RILs) derived from Jing711 (contains 17 subunit on Glu-B1x) and Pm97034 (contains 14 subunit on Glu-B1x) were used to validate the accuracy of the primers, which showed that the two specific markers could be used together to distinguish alleles at Glu-B1x locus and accelerate wheat quality breeding by marker assisted selection.