Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.     

Deletional Protein Engineering Based on Stable Fold

Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.     

The peripheral chimerism of bone marrow–derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice

Bone marrow–derived cells represent a heterogeneous cell population containing haematopoietic stem and progenitor cells. These cells have been identified as potential candidates for use in cell therapy for the regeneration of damaged tissues caused by trauma, degenerative diseases, ischaemia and inflammation or cancer treatment. In our study, we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP⁺) transplanted cells into non-haematopoietic tissues. We investigated the repair of damage to the BM, peripheral blood, spleen and thymus and assessed the ability of this treatment to induce the entry of BM cells or GFP⁺lin⁻Sca-1⁺ cells into non-haematopoietic tissues. The transplantation of BM cells or GFP⁺lin⁻Sca-1⁺ cells from GFP transgenic mice successfully repopulated haematopoiesis and the haematopoietic niche in haematopoietic tissues, specifically the BM, spleen and thymus. The transplanted GFP⁺ cells also entered the gastrointestinal tract (GIT) following whole-body irradiation. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and induced the entry of transplanted cells into the small intestine and liver. This result demonstrates that grafted BM cells or GFP⁺lin⁻Sca-1⁺ cells are not transient in the GIT. Thus, these transplanted cells could be used for the long-term treatment of various pathologies or as a one-time treatment option if myeloablation-induced chimerism alone is not sufficient to induce the entry of transplanted cells into non-haematopoietic tissues.     

Surface treated Pteris vittata L. pinnae powder used as an efficient biosorbent of Pb(II), Cd(II), and Cr(VI) from aqueous solution

Biosorption is a surface-dependent phenomenon. Surface modifications by chemical treatment methods could either improve or reduce the biosorption capacity of potential biosorbents. In the present work, pristine Pteris vittata L. pinnae (PPV) powder was treated separately with sodium hydroxide (NaOH), calcium chloride (CaCl₂), and nitric acid (HNO₃). The pristine and treated biosorbents were used to assess the biosorption of Pb(II), Cd(II), and Cr(VI) as a function of pH. Kinetics and adsorption isotherms were studied. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and scanning electron microscope combined with energy dispersive x-ray (SEM-EDX) spectroscopic techniques were used to characterize the biosorbents before and after chemical treatments. The possible functional groups contributing to the metal sorption were identified. Results revealed favorable biosorption of Pb(II), Cd(II), and Cr(VI) described by pseudo-second order kinetics. NaOH-treated P. vittata (NPV) showed higher biosorption capacity for Pb(II) and Cd(II) compared to that of PPV. ATR-FTIR studies indicated that -OH, -COOH, and -NH₂ groups were mainly involved in Cr(VI) and -OH in Pb(II) and Cd(II) biosorption. The enhanced efficiency of NPV and CaCl₂ treated P. vittata (CPV) in the uptake of Pb(II) and Cd(II) compared to PPV can be associated with their altered physicochemical characters.     

Characterization of grain protein content gene (<Emphasis Type="Italic">GPC</Emphasis>-<Emphasis Type="Italic">B1</Emphasis>) introgression lines and its potential use in breeding for enhanced grain zinc and iron concentration in spring wheat

At least two billion people around the world suffer from micronutrient deficiency, or hidden hunger, which is characterized by iron-deficiency anemia, vitamin A and zinc deficiency. As a key staple food crop, wheat provides 20% of the world’s dietary energy and protein, therefore wheat is an ideal vehicle for biofortification. Developing biofortified wheat varieties with genetically enhanced levels of grain zinc (Zn) and iron (Fe) concentrations, and protein content provides a cost-effective and sustainable solution to the resource-poor wheat consumers. Large genetic variation for Fe and Zn were found in the primitive and wild relatives of wheat, the potential high Zn and Fe containing genetic resources were used as progenitors to breed high-yielding biofortified wheat varieties with 30–40% higher Zn content. Grain protein content (GPC) determines processing and end-use quality of wheat for making diverse food products. The GPC-B1 allele from Triticum turgidum L. var. dicoccoides have been well characterized for the increase in GPC and the associated pleiotropic effect on grain Zn and Fe concentrations in wheat. In this study effect of GPC-B1 allele on grain Zn and Fe concentrations in wheat were measured in different genetic backgrounds and two different agronomic management practices (with- and without foliar Zn fertilization). Six pairs of near-isogenic lines differing for GPC-B1 gene evaluated at CIMMYT, Mexico showed that GPC-B1 influenced marginal increase for grain Zn, Fe concentrations, grain protein content and slight reduction in kernel weight and grain yield. However, the magnitude of GPC and grain Zn and Fe reductions varied depending on the genetic background. Introgression of GPC-B1 functional allele in combination with normal or delayed maturity alleles in the CIMMYT elite wheat germplasm has the potential to improve GPC and grain Zn and Fe concentrations without the negative effect on grain yield due to early senescence and accelerated maturity.     

Mountain Aspect Influences the Genetic Clustering of Psychrotolerant Phosphate Solubilizing <Emphasis Type="Italic">Pseudomonads</Emphasis> in the Uttarakhand Himalayas

Fourteen cold tolerant phosphate solubilizing bacteria isolated from high altitude representative locations of the two major mountain aspects of the Uttarakhand Himalayas (cooler north and warmer south facing slopes) were selected for this study. The tricalcium phosphate (TCP) solubilizing abilities of the isolates were estimated at three different incubation temperatures viz., 4, 15, and 30°C under in vitro conditions. Irrespective of their geographical origin, all the isolates recorded maximum P release values at 30°C. At 4°C, the isolates from the north facing slope were found to release significantly higher levels of P, as compared to the isolates from the south facing slopes. Alternatively at 15°C, the isolates from the south facing slope were found to release significantly higher levels of P. Initial confirmation of their genus level identity as Pseudomonads was arrived by amplification of a 990 bp fragment of the 16S rRNA gene using genus specific primers. Further putative species level identification was arrived by sequencing of the 16S rRNA gene. The diversity among the isolates was determined by rep-PCR using the primers BOX, ERIC, and ERIC2. A composite dendrogram constructed using the rep-PCR profiles revealed that the isolates from the north and south mountain aspects formed separate major clusters. The extent of diversity was greater among the isolates from the south mountain aspect. This study reveals the potential of rep-PCR in determining the genetic diversity among Pseudomonads selected for a single functional trait, but varying in their geographical origin.     

Phosphate solubilization and growth promotion by <Emphasis Type="Italic">Pseudomonas fragi</Emphasis> CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere

Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude garlic rhizosphere from the Indian Himalayas, are reported here. The identity of the isolate was arrived on the basis of its biochemical features and sequencing of the 16S rRNA gene. The isolate grew and solubilized phosphate at temperatures ranging from 4 to 30°C. Besides solubilizing P it produced indole acetic acid (IAA) and hydrogen cyanide (HCN). Seed bacterization with the isolate significantly increased the percent germination, rate of germination, plant biomass and nutrient uptake of wheat seedlings. While Pseudomonas fragi is normally associated with the spoilage of dairy products stored at cold temperatures, this is an early report on the plant growth promoting ability of the bacterium.     

KINALYZER, a computer program for reconstructing sibling groups

A software suite KINALYZER reconstructs full-sibling groups without parental information using data from codominant marker loci such as microsatellites. KINALYZER utilizes a new algorithm for sibling reconstruction in diploid organisms based on combinatorial optimization. KINALYZER makes use of a Minimum 2-Allele Set Cover approach based on Mendelian inheritance rules and finds the smallest number of sibling groups that contain all the individuals in the sample. Also available is a 'Greedy Consensus' approach that reconstructs sibgroups using subsets of loci and finds the consensus of the partial solutions. Unlike likelihood methods for sibling reconstruction, KINALYZER does not require information about population allele frequencies and it makes no assumptions regarding the mating system of the species. KINALYZER is freely available as a web-based service.     

Isolation and Characterization of A Metal-resistant Pseudomonas Aeruginosa Strain

Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin.