Isoelectric point (pI)-based phase separation (pI-BPS) purification of elastin-like polypeptides (ELPs) containing charged,biologically active fusion proteins (ELP-FPs)

Elastin-like polypeptides (ELPs) are peptide-based biomaterials with residue sequence (VPGXG)n where X is any residue except proline. ELPs are a useful modality for delivering biologically active proteins (growth factors, protease inhibitors, anti-inflammatory peptides, etc.) as fusion proteins (ELP-FP). ELP-FPs are particularly cost-effective because they can be rapidly purified using Inverse Temperature Cycling (ITC) via the reversible formation and precipitation of entropically driven aggregates above a transition temperature (T_t). When ELP fusion proteins (ELP-FPs) contain significant charge density at physiological pH, electrostatic repulsion between them severely inhibits aggregate formation. The literature does not currently describe methods for purifying ELP-FPs containing charged proteins on either side of the ELP sequence as fusion partners without organic solvents. Here, the isoelectric point (pI) of ELP-FPs is discussed as a means of neutralizing surface charges on ELP-FPs and increasing ITC yield to dramatically high levels. We use pI-based phase separation (pI-BPS) to purify ELP-FPs containing cationic and anionic fusion proteins. We report a dramatic increase in protein yield when using pI-BPS for purification of ELP-FPs. Proteins purified by this method also retain the functional activity of the protein present in the ELP-FP. Techniques developed here enable significant diversification of possible fusion proteins delivered by ELPs as ELP-FPs by allowing them to be produced and purified at higher quantities and yields.     

Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(?)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.     

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n = 18) were split into five aliquots for dilution (37 °C; 50 × 10⁶ spermatozoa ml^?1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4 °C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN₂ level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4 h post-thawing (37 °C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p < 0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n = 2) spermatozoa was higher (p < 0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.     

Isolation of antibacterial compounds from Quercus dilatata L. through bioassay guided fractionation

Background

Four medicinal plants (Chrozophora hierosolymitana Spreng, Chrysanthemum leucanthemum L., Ephedra gerardiana Wall. ex Stapf, and Quercus dilatata L.) used by indigenous healers to treat various infectious diseases were selected for the present study. The major objective of the present study was isolation and characterization of antimicrobial components from the crude plant extracts using bioassay guided fractionation.

Methods

Seven methanolic extracts of the four plants were screened to identify any antimicrobial agents present in them. The active crude plant extract was fractionated first by solvent partitioning and then by HPLC. Characterization of the active fractions was done by using spectrophotometer.

Results

All the seven methanolic extracts showed low antifungal activity, however, when these extracts were tested for antibacterial activity, significant activity was exhibited by two extracts. The extract of aerial parts of Q. dilatata was most active and therefore, was selected for further analysis. Initially fractionation was done by solvent-solvent partitioning and out of six partitioned fractions, ethanol fraction was selected on the basis of results of antibacterial activity and phytochemical analysis. Further, fractionation was carried out by RP- HPLC and purified active subfractions were characterized by comparing their absorption spectra with that of the known natural products isolated from the plants of Quercus genus.

Discussion and conclusion

The results suggest that this is the first report of the isolated antibacterial compounds from this genus.     

In Planta Transformation of Tomato

In this study, in planta transformation of tomato (Solanum lycopersicum L.), using fruit injection and floral dip, is reported. Agrobacterium tumefaciens strain EHA 105 containing one of three constructs, i.e., pROKIIAP1GUSint (carrying the Apetala 1 [AP1] gene), pROKIILFYGUSint (carrying the LEAFY [LFY] gene), or p35SGUSint (carrying the β-glucuronidase [GUS] gene), was used for plant transformation. For fruit injection transformation, no significant effects (p > 0.05) of the construct used were observed. The highest frequency of transformation was obtained following 48-h incubation of tomato fruit with bacterial cells harboring either one of the three constructs; transformation frequencies of 17%, 19%, and 21% for AP1, LFY, and GUS gene constructs, respectively, were obtained. When fruit maturity was evaluated in fruit injection experiments, mature red fruit resulted in higher frequency of transformants than immature green fruit with 40%, 35%, and 42% for AP1, LFY, and GUS gene constructs, respectively. For floral dip transformation, a higher number of transformants was obtained when the GUS gene construct was used instead of either the AP1 or LFY gene construct, thus suggesting a possible inhibitory effect of the flowering genes used. When flowers were transformed prior to rather than following pollination, they yielded a higher transformation frequency, 12% for the LFY construct and 23% for the GUS construct (p < 0.05), although no transformant was obtained with the AP1 gene construct. All putative GUS-positive transformants were analyzed using polymerase chain reaction and confirmed for the presence of the transgene. Compared to control plants, transgenic plants carrying either the AP1 or LFY transgene flowered earlier and showed several different morphological characters.     

Genetic modification of Gossypium arboreum universal stress protein (GUSP1) improves drought tolerance in transgenic cotton (Gossypium hirsutum)

Cotton crop suffers shortage of irrigation water at reproductive stage which reduces the yield and fibre quality. Universal stress proteins belong to Pfam00582 which enables several plants to cope with multiple stresses via ATP binding. GUSP1 (Gossypium arboreum USP) is one of such proteins; its amino acids were mutated after in silico simulations including homology modeling and molecular docking analysis. Transgenic cotton plants were developed through Agrobacterium mediated genetic transformation by using mutated pmGP1 and non mutated pGP1 constructs under CaMV35S promoter. PCR and semi-quantitative PCR analyses confirmed the amplification and expression of transgene in transgenic plants. It was revealed that leaf relative water content, total chlorophyll content, CO₂ assimilation as net photosynthesis, stomatal conductance, total soluble sugars and proline content was significantly increased at P ≤ 0.0001 and P ≤ 0.001 in both the pmGP1 and pGP1 transgenic plants as compared to non transgenic control plants. Moreover, relative membrane permeability and the transpiration rate were reduced significantly at P ≤ 0.0001 and P ≤ 0.001 respectively in transgenic plants under drought stress. Furthermore, the T1 transgenic seedlings containing pmGP1 mutated construct showed longer roots under desiccation stress imposed by 5% PEG. Transgene inheritance into the T1 progeny plants was confirmed by amplification through PCR and integration through Southern blot. Hence, our results pave the way to utilize the mutagenized known genes for increasing endurance of plants under drought stress. This will help to increase our understanding of drought tolerance/ sensitivity in cotton plants at the molecular level.Supplementary Information The online version contains supplementary material available at 10.1007/s12298-021-01048-5.     

An efficient protocol for transient transformation of intact fruit and transgene expression inCitrus

In this study, conditions were optimized for transient gene expression in Rough Lemon (Citrus jambhiri Lush.), a major rootstock used in the citrus growing regions of Pakistan.Agrobacterium tumefaciens carrying the binary vector p35GUSINT, containingNPT II andGUS genes, was used in the study. The transformation method was based on injection ofAgrobacterium intoCitrus fruits followed by histochemical assay ofGUS activity in different tissues. Different tissues of mature fruits exhibited significantly different percentages of transientGUS expression: in rind (76%), spongy tissue (92%), juice vesicles (0%) and seeds (83%) (P<0.01)., The incubation period after injecting theAgrobacterium culture also showed a significant (P<0.01) effect on the transient expression ofGUS in these tissues. An incubation period of 48 h was found to be the best (72%) for transformation of whole fruit, followed by 72 h (67%) and 96 h (49%). TransientGUS expression also varied significantly (P<0.01) in juice vesicles and seeds as fruit matured. Juice vesicles from mature fruits showed no transientGUS expression, while those from immature fruits showed 50% expression. Furthermore, transformation of seeds had no effect on their germination capability. Germinating seeds from mature fruits injected withAgrobacterium culture showed tolerance to kanamycin (100 mg/L), which varied with the incubation period (55% at 48 h, 25% at 72 h and 23% at 96 h). This report offers an easy protocol for transient expression studies of transgenes and has the potential to be used for stable transformation ofCitrus.     

Metabolic signatures altered by in vitro temperature stress in <Emphasis Type="Italic">Ajuga bracteosa</Emphasis> Wall. ex. Benth.

To elucidate how biosynthesis of plant metabolites is affected by temperature, metabolite profiles from in vitro regenerated plants raised under different temperature regimes of 10, 15 °C, 20 °C, 25 °C and 30 °C were obtained using electrospray ionization mass spectrometry (ESI-MS), and principal component analysis (PCA) was carried out to identify key metabolites. Several bin masses were detected by PCA loading scatter plots which separated the samples. In-house bin program selectively manifested the putative known metabolites depending on % total ions count and intensity of selected bins in the plant samples. Total phenolic and flavonoid content were harvested to highest levels (12.9 mg GAE/g DW and 9.3 mg QE/g DW), respectively, at 15 °C. Besides, pinoresinol (lignan), some of the vital amino acids such as serine, methionine, histidine and glutamine were found to be at higher amount in plants raised at 15 °C. Significant phenylpropanoids like cinnamic acid, caffeic acid and quercitol were detected at a higher concentration in plants raised at 15 °C as compared to other treatments. However, phosphoenolpyruvate, and oxalosuccinate (intermediates of the pentose phosphate pathway) were accumulated the most in plants raised at 30 °C and they were detected with lowest values at 10 °C. Glucose and deoxy-xylose 5 phosphate (intermediates of TCA cycle) were found in higher amounts at temperature treatments of 15 and 25 °C, respectively. We conclude that a low-temperature treatment (15 °C) results in a stress-induced accumulation of a variety of pharmacologically important secondary metabolites.