Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 µg/g), chlorogenic acid (0.0978 ± 0.0046 µg/g), and quercetin (0.0971 ± 0.0046 µg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy.

BACKGROUND: Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern. METHODS: Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy. RESULTS: Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation. CONCLUSIONS: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein. GENERAL SIGNIFICANCE: Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.

Cis-acting motifs in artificially synthesized expression cassette leads to enhanced transgene expression in tomato (Solanum lycopersicum L.).

Efficacy of artificial synthetic expression modules was compared with native CaMV35S and DECaMV35S promoter in transgenic tomato developed by Agrobacterium-mediated transformation. The promoters under trial were CaMV35S-mec (PcamI), CaMV35S (PcamII), DECaMV35S (PcamIII), synthetic minimal expression cassette (Pmec), complete expression cassette (Pcec), double enhancer expression cassette (Pdec) and triple enhancer expression cassette (Ptec) for driving the uidA gene for ß-glucuronidase (GUS) activity. The promoter efficiency based on average of GUS expression in T(0) and T(1) transgenic tomato was in the order Pcec > Pdec > PcamIII > PcamII > PcamI > Ptec > Pmec. The two promoters Pcec and PcamIII were deployed for development of insect-resistant transgenic tomato with optimal expression of modified cry1Ac insecticidal toxin gene from Bacillus thuringiensis (Bt). The transgenic status and copy number of the cry1Ac in T(0) transgenic tomato was confirmed through PCR, Southern hybridization, RT-PCR and Western immunoassay, while toxin expression was monitored by DAS-ELISA. The expression level of Cry1Ac toxin driven by Pcec in T(0) population ranged from 0.08 to 0.8% of total soluble protein (TSP) that was significantly higher to PcamIII which ranged from 0.02 to 0.13% of TSP. The outcome of insect mortality bioassay with Helicoverpa armigera correlated well with the results of DAS-ELISA. The higher expression of cry1Ac gene driven by Pcec promoter in transgenic tomato did not show any yield penalty and reflected complete protection, while low recovery of promising transgenics expressing Cry1Ac toxin driven by PcamIII was a major limitation for complete protection against the fruit borer insect.

Differential subcellular targeting of recombinant human α1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants.

The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human proteinase inhibitor (α1-PI) was analyzed via targeting to different subcellular locations, like endoplasmic reticulum (ER), apoplast, vacuole and cytosol in leaves of transgenic tomato plants. In situ localization of the recombinant α1-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant α1-PI in T0 and T1 transgenic tomato plants was achieved from 1.5 to 3.2% of total soluble protein (TSP) by retention in ER lumen, followed by vacuole and apoplast, whereas cytosolic targeting resulted into degradation of the protein. The plant-derived recombinant α1-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant α1-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ∼48-50 kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant α1-PI revealed the structural integrity of the recombinant protein comparable to native serum α1-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant α1-PI showed compartment-specific N-glycosylation of the protein targeted to ER, apoplast and vacuole. Conformational studies based on urea-induced denaturation and circular dichroism (CD) spectroscopy revealed relatively lower stability of the recombinant α1-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified α1-PI in rat, by intravenous route, revealed significantly faster plasma clearance and lower area under curve (AUC) of recombinant protein. Our data suggested significance of protein sorting sequences and feasibility to use transgenic plants for the production of stable, glycosylated and biologically active recombinant α1-PI for further therapeutic applications.

Computational tridimensional protein modeling of Cry1Ab19 toxin from Bacillus thuringiensis BtX-2.

We report the computational structural simulation of the Cry1Ab19 toxin molecule from B. thuringiensis BtX-2 based on the structure of Cry1Aa1 deduced by x-ray diffraction. Validation results showed that 93.5% of modeled residues are folded in a favorable orientation with a total energy Z-score of -8.32, and the constructed model has an RMSD of only 1.13. The major differences in the presented model are longer loop lengths and shortened sheet components. The overall result supports the hierarchical three-domain structural hypothesis of Cry toxins and will help in better understanding the structural variation within the Cry toxin family along with facilitating the design of domain-swapping experiments aimed at improving the toxicity of native toxins.

High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding ß-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 µM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

Expression and purification of recombinant human alpha1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity.

Human alpha(1)-proteinase inhibitor (alpha(1)-PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant alpha(1)-PI has great potential in therapeutic applications. We report here the expression of a synthetic alpha(1)-PI gene and its variants in Escherichia coli. Modified alpha(1)-PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant alpha(1)-PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant alpha(1)-PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7-9 mg l(-1) of bacterial culture (approximately 5.2 g wet cell mass). E. coli expressed recombinant alpha(1)-PI showed specific anti-elastase activity and appeared as a single band of approximately 45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant alpha(1)-PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified alpha(1)-PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active alpha(1)-PI and its variants in E. coli for further therapeutic applications.

Effect of point mutations in translation initiation context on the expression of recombinant human alpha(1)-proteinase inhibitor in transgenic tomato plants.

The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human alpha(1)-proteinase inhibitor (alpha(1)-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant alpha(1)-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at -3 position eliminated most of the alpha(1)-PI expression, while mutation at -3 alone resulted in about sevenfold reduction. The presence of steady-state levels of alpha(1)-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.

Expression of modified gene encoding functional human alpha-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.

Glutamine synthetase from N2-fixing cyanobacterium Nostoc muscorum--purification, biochemical and immunological characteristics.

A modified procedure for purification of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming)] from N2-fixing cyanobacterium N. muscorum, to homogeneity is described using DEAE-Sephadex and Blue-Sepharose affinity chromatography. Specific activities of the purified enzyme in biosynthetic and transferase assays were 8.5 and 28 mumole product formed min-1 mg-1 protein. Apparent molecular mass of native GS enzyme was about 610 kDa as estimated by gel filtration. On SDS-PAGE the enzyme protein migrated as single band with molecular weight of 51 kDa. Apparent Michaelis Menten constant (Km) for glutamate, glutamine, ATP and ammonium were 2.8, 4.0, 0.35 and 0.82 mM respectively. Ammonium and structural analogues of glutamine and ammonium viz, methionine sulfone (MSO), methionine-DL-sulfoximine (MSX), ethylenediamine (EDA) and glyphosine significantly inactivated the enzyme activity while azaserine showed partial inhibition. Polyclonal antibodies raised against purified GS protein of N. muscorum showed high specificity to both crude and pure GS preparations in different immunological assays like double diffusion, rocket immunoelectrophoresis, ELISA and western blotting. With serological procedure a microquantity of GS-antigen upto 2 ng in vivo and immunological relationship of the protein with different strains have been documented.

Drift in ultraviolet sensitivity and expression of mutations during synchronous growth of cyanobacterium Anacystis nidulans.

Synchrony with respect to cell division and DNA synthesis in cultures of Anacystis nidulans was induced by a light-dark-light regimen. At periodic intervals in the cell-division cycle, DNA, RNA, protein contents, UV sensitivity and induction of mutations were assayed. The DNA, RNA and protein syntheses were periodic and reached maximal values before the separation of cells. The DNA content started to increase at about the 5th hour and doubled at about the 13th hour followed by a plateau of 4-6 h. Wild-type A. nidulans was highly sensitive to UV radiation during the period showing an increase in cell number (rise phase) and the early part of DNA synthesis (synthetic phase). Significant resistance to UV, however, developed in the later stage of the DNA synthesis. This resistance decreased considerably during the next rise phase. On the other hand, in a UV-sensitive strain of A. nidulans (uvs67) there was no appreciable change in the UV sensitivity during the cell-division and DNA-synthesis phases. Induction of mutation frequency patterns of all the markers (fil, blu, yel, vir, nit, strR) in the wild-type showed a short initial lag followed by an abrupt increase resulting in a peak of mutation frequency in the early part of DNA synthesis and subsequently a second plateau. The induction of mutation frequencies in the uvs67 strain was comparatively low and remained constant throughout the cell division cycle. These results suggest the possibilities of an error-prone dark repair and a stringent relationship between DNA replication and repair of UV damage for expression of mutations in cyanobacterium A. nidulans.

Mutagenesis of free and intracellular cyanophage AS-1 by ultraviolet, N-methanyl-N'-nitro-N-nitrosoguanidine and acriflavine.

Mutagenic actions of ultraviolet irradiation (UV), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and acriflavine (photodynamic) were tested in free and intracellular phage AS-1 infecting Anacystis nidulans IU625. Spontaneous and induced mutations with particular reference to host range (h) and minute plaque formation (m) were investigated. The spontaneous mutation frequencies varied from 10(-9) to 10(-8) and from 2 X 10(-5) to 2 X 10(-4) for h and m mutants respectively. UV was efficient in inducing h and m markers in free phage particles as well as intracellular phage; MNNG induced both markers in intracellular phage only, and acriflavine induced m mutants only in free as well as in infecting phages. UV-induced mutations in free phage were photo-reactivable by visible light. With all the mutagens used, maximal induction of both markers was observed with treatment of 2-h complexes.

Photoreactivation of ultraviolet irradiated blue-green alga: Anacystis nidulans and cyanophage AS-1.

Ultraviolet (UV) inactivation and photoreactivation of Anacystis nidulans and cyanophage AS-1 was studied at different wavelengths. UV inactivation of free phage particles and one and two hour host-phage complexes (intracellular phages) were exponential. UV resistance of plaque forming units was attained at the latter phase of latent period. Black, blue and white lights were able to photoreactivate the UV irradiated A. nidulans whereas green, yellow and red lights were not. However, incubation of A. nidulans for more than 2 hours in black light resulted in loss of viability but shift to red light caused significant recovery. This suggests the involvement of two types of photoreactivation, i.e. of photoenzymatic repair of DNA and of the repair of the photosynthetic apparatus of A. nidulans.