D. V. Amla

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.

Changes in protein pattern during different developmental stages of somatic embryos in chickpea

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg−1(f.m.)] to cotyledonary stage [4.8 μg mg−1(f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg−1(f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.

Retraction Note: 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 Agrobacteriumtumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD600 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 T0 transgenic plants were generated, representing 3.6% transformation frequency. T0 transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T1 progeny. PCR, RT-PCR, and Southern hybridization analysis of T0 and T1 transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T0 and T1 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.

Effect of point mutations in translation initiation context on the expression of recombinant human α1-proteinase inhibitor in transgenic tomato plants

Abstract The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human α1-proteinase inhibitor (α1-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 α1-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 α1-PI expression, while mutation at −3 alone resulted in about sevenfold reduction. The presence of steady-state levels of α1-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 and purification of recombinant human α1-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.

Molecular Characteristics of glnA Linked Mutations in the Nitrogen-Fixing Cyanobacterium Nostoc muscorum

The glutamine synthetase (GS)-impaired mutants of Nostoc muscorum, isolated as ethylenediamine (EDA) resistant phenotype, were characterized for glnA linked processes to investigate the molecular mechanism of GS impairment. The GS enzyme was purified to homogeneity from the wild and mutant strains of N. muscorum employing ion-exchange followed by affinity chromatography. The final yield and specific activity of the enzyme were low in the mutant strains as compared to the wild type. The molecular weight of the native enzyme and the subunit size of GS polypeptide in all the strains were identical. Compared to the wild type, the GS purified from the mutant strains showed refractory response to its structural analogues like methionine sulfone (MSO), methionine sulfoximine (MSX), EDA, and metabolic substrates, such as ammonium and glutamine. The Km and Vmax for the substrates of transferase and biosynthetic reactions were altered in the mutant strains. The immunological assays performed with partially purified IgG molecules raised against wild-type GS revealed quantitative and qualitative heterogeneity for cross reactivity among the wild-type and EDA-resistant strains. Southern hybridization of genomic DNA in all the strains of N. muscorum restricted with different enzymes showed identical pattern with Anabaena 7120 glnA probe and localization of complete gene for GS on 7.5 kb HindIII fragment. Northern blot analysis indicated differential levels of GS specific mRNA and abundance of glnA transcripts of 1.7 and 1.9 kb in the mutant strains. Results suggested that point mutation(s) inflicted in glnA gene of N. muscorum are responsible for differential production of catalytically modified GS in EDA-resistant phenotypes.

Identification and Quantification of Heterologous Compounds Parthenin and Organic Acids in Parthenium Hysterophorus L. Using HPLC-PDA-MS-MS

Parthenium hysterophorus L., is an obnoxious weed known for its environmental health hazards and medicinal uses. These characteristics are due to presence of sesquiterpene lactones and organic acids; therefore a rapid and sensitive analytical procedure using HPLC-PDA-MS-MS was developed and optimized for separation, identification, and quantification of parthenin and six organic acids. Separation and characterization of compounds was achieved on a RP-C18 column with 1% acetic acid in water (A) and acetonitrile (B) as a mobile phase at a flow rate of 0.6 mL min−1 and by matching their UV and mass spectra with reference compounds. Six organic acids (ferulic acid, 0.1 mg g−1 to coumaric acid, 13.6 mg g−1) and parthenin (27.4 mg g−1) were characterized within 26 minutes of chromatographic separation in plant extract. The calibration curves are linear with correlation coefficients from 0.985 to 0.998, limit of detection and quantification ranged between 1.0 µg mL−1 (anisic acid) to 2.2 µg mL−1 (parthenin) and 2.5 µg mL−1 (coumaric acid) to 5.2 µ g mL−1 (parthenin) and recovery ranged between 90.9% to 97.3%. To the best of our knowledge this is the first report for the simultaneous separation of parthenin and organic acids. The method is applicable for screening of commercial crops, medicinal plants, and their products which might be mixed with P. hysterophorus during harvesting period.

Physiological and biochemical analysis of the glutamine synthetase-impaired mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Three types of glutamine synthetase (GS)-impaired mutants (gln) ofNostoc muscorum were isolated as ethylenediamine (EDA)-resistant phenotypes and characterized with respect to heterocyst development, nitrogen fixation, ammonium metabolism, photosynthetic characteristics, and glutamine synthetase activity. The criterion for categorizing the mutants was the extent of loss of GS activity (both in transferase and biosynthetic assays) compared with wild type; it was 70% in EDA-1, 30% in EDA-2, and more than 90% in EDA-3 strains. The level of nitrogenase activity in mutant strains was proportionate to heterocyst frequency and was found refractory to ammonium and EDA repression. In EDA-resistant strains, development of heterocysts and their spacing pattern remained unaffected and did not respond to treatment of amino acid analogues, drugs, and ammoniacal compounds which otherwise either stimulated or suppressed the number and altered the spacing pattern in wild type. A biphasic pattern of ammonium uptake indicating two transport systems was observed in all the strains except that the Km values for both high- and low-affinity systems were altered in mutant strains. In vivo treatment with MSX or EDA significantly inhibited the GS activity in wild type, whereas mutant strains did not respond to these treatments and were able to liberate NH4+ continuously into the medium without MSX treatment. During NH4+ uptake, percentage inhibition of O2 evolution and changes in increase of fluorescence intensity were low in EDA strains compared with wild type. Assessment of GS protein with antibodies against GS and quantitative polyacrylamide gel electrophoresis (PAGE) suggested that loss in specific activity of GS per milligram of extractable protein in EDA mutants was owing to low production of GS-specific protein. SDS-PAGE of purified GS enzyme from all the strains revealed only one polypeptide band of molecular weight of about 51.28 kDa.

Characterization and functional validation of two scaffold attachment regions (SARs) from Cicer arietinum (L.)

A library of SARs has been prepared from chickpea by Illumina sequencing of DNA fragments that co-isolate with the nuclear scaffold by lithium diiodosalicylate treatment. Seven fragments were screened on the basis of SAR associated motifs and their interactions were tested with the nuclear scaffold of chickpea by in vitro binding assay. SAR 1 and SAR 2 bind strongly in comparison to other SARs to the nuclear scaffold of chickpea and tomato during in vitro binding assay. To investigate the effect of SARs on transgene expression and variation among transformants, NBRI 1.1 (harbouring GUS expression cassette with single SAR1 fragment), NBRI 2.1 (with single SAR2 fragment) and NBRI 1.2 (with two SAR1 fragments) vectors were prepared for plant transformation and transgenic tomato plants were developed, plants transformed with pBI121 functioned as a control. The enzymatic activity of GUS increased 9.52 fold in NBRI 2.1, 17.82 fold in NBRI 1.1 and 51.4 fold increase in NBRI 1.2 in comparison to pBI121. Chickpea was transformed with NBRI 1.2 and pBI121, where GUS enzymatic activity increased 13.789 fold in NBRI 1.2 in comparison to pBI121.

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.