The affinity concept in bioseparation: evolving paradigms and expanding range of applications

The meaning of the word affinity in the context of protein separation has undergone evolutionary changes over the years. The exploitation of molecular recognition phenomenon is no longer limited to affinity chromatography modes. Affinity based separations today include precipitation, membrane based purification and two-phase/three-phase extractions. Apart from the affinity ligands, which have biological relationship (in vivo) with the target protein, a variety of other ligands are now used in the affinity based separations. These include dyes, chelated metal ions, peptides obtained by phage display technology, combinatorial synthesis, ribosome display methods and by systematic evolution of ligands by exponential enrichment (SELEX). Molecular modeling techniques have also facilitated the designing of biomimetic ligands. Fusion proteins obtained by recombinatorial methods have emerged as a powerful approach in bioseparation. Overexpression in E. coli often result in inactive and insoluble inclusion bodies. A number of interesting approaches are used for simultaneous refolding and purification in such cases. Proteomics also needs affinity chromatography to reduce the complexity of the system before analysis by electrophoresis and mass spectrometry are made. At industrial level, validation, biosafety and process hygiene are also important aspects. This overview looks at these evolving paradigms and various strategies which utilize affinity phenomenon for protein separations.     

In silico modeling and hydrogen peroxide binding study of rice catalase

Homology modeling of the catalase, CatC cloned and sequenced from rice (Oryza sativa L., cv Ratna an Indica cultivar) has been performed based on the crystal structure of the catalase CatF (PDB code 1m7s) by using the software MODELLER. With the aid of molecular mechanics and molecular dynamics methods, the final model is obtained and is further assessed by PROCHECK and VERIFY - 3D graph, which show that the final refined model is reliable. With this model, a flexible docking study with the hydrogen peroxide, the substrate for catalase, is performed and the results indicate that Arg310, Asp343 and Arg346 in catalase are three important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate. These hydrogen-bonding interactions play an important role for the stability of the complex. Our results may be helpful for further experimental investigations.     

A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency

A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/Km value (Vmax/Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 degrees C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.     

Effects of murine endotoxemia on lymphocyte subsets and clearance of staphylococcal pulmonary infection

In a model of staphylococcal pneumonia initiated during systemic endotoxemia in BALB/c mice, a significant reduction of the number of circulating CD4+ and CD8+ T-lymphocytes, B-lymphocytes, and NK cells, as well as lung-resident total T- and CD4+ T-lymphocytes was demonstrated. Staphylococcus aureus exposure only induced a similar decrease of lymphocyte subsets in the blood. However, the number of lung-resident total T- and CD4+ T-lymphocytes was increased. More viable bacteria were recovered from the lungs of S. aureus-infected mice than from those animals previously treated with lipopolysaccharide (LPS) followed by a staphylococcal challenge. These results indicate that LPS-induced reduction in the number of circulating lymphocyte subsets and lung-resident total T- and CD4+ T-lymphocytes do not increase susceptibility to staphylococcal respiratory infection. Moreover, LPS challenge prior to S. aureus exposure significantly improves clearance of the bacteria in the lung.     

MiRANN: a reliable approach for improved classification of precursor microRNA using Artificial Neural Network model

MicroRNA (miRNA) is a special class of short noncoding RNA that serves pivotal function of regulating gene expression. The computational prediction of new miRNA candidates involves various methods such as learning methods and methods using expression data. This article has proposed a reliable model - miRANN which is a supervised machine learning approach. MiRANN used known pre-miRNAs as positive set and a novel negative set from human CDS regions. The number of known miRNAs is now huge and diversified that could cover almost all characteristics of unknown miRNAs which increases the quality of the result (99.9% accuracy, 99.8% sensitivity, 100% specificity) and provides a more reliable prediction. MiRANN performs better than other state-of-the-art approaches and declares to be the most potential tool to predict novel miRNAs. We have also tested our result using a previous negative set. MiRANN, opens new ground using ANN for predicting pre-miRNAs with a promise of better performance.     

Haptoglobin polymorphism among fourteen populations of India

Haptoglobin (HP) is a serum protein that has the capability of binding the extracorpuscular haemoglobin released during haemolysis. It plays an important role in protection of haemolytic disease by reducing the oxidative and peroxidative potential at free haemoglobin. The present study was aimed to determine the prevalence of HP polymorphism among different Indian populations, anthropologically belonging to diverse ethnicity. The polymorphism was screened among 642 unrelated individuals belonging to 14 population groups of India including both tribal and non-tribal caste groups from different geographical regions of India with distinct linguistic affiliations. An attempt is also made to understand the distribution of HP polymorphism among the studied populations. The result reveals the HP gene to be polymorphic in all the studied populations. Except the two tribal populations (Thotis of Andhra Pradesh and Patelias of Rajasthan) and one caste population (Rajput of Himachal Pradesh), all the studied populations are found to obey the Hardy-Weinberg equilibrium. The significance of the present study is elucidated with the prevalence of high mutant HP*2 allele frequency in India. Selection could be one of the most plausible explanations for this high HP frequency because of its uniformly high occurrence among all the studied populations.     

Carbohydrate content and antioxidative potential of the seed of three edible indica rice (Oryza sativa L.) cultivars

Rice (Oryza sativa L.) grains or seeds are known to lose much of their nutrient and antioxidant contents, following polishing. The current study was undertaken to evaluate and compare the carbohydrate content and antioxidant parameters in the unpolished and polished seeds of three edible indica rice cultivars, namely Swarna (SW), the most popular indica rice cultivar in India and aromatic or scented cultivars Gobindobhog (GB) and Pusa Basmati (PB). While both the sucrose and starch content was the maximum in PB seeds (both unpolished and polished), the amylose content was the highest in SW polished seeds. SW polished seeds were superior as compared to GB and PB cultivars in terms of total antioxidant capacity, DPPH radical scavenging and Fe(II) chelation potential, as well as the highest lipoxygenase (LOX) inhibition or H2O2 scavenging potential, probably due to the maximum accumulation of total phenolics and flavonoids, the two important antioxidants. The reducing power ability was, however, identical in both SW and GB polished seeds. The PB polished seeds were more potent in superoxide and hydroxyl scavenging, whereas GB in nitric oxide (NO) scavenging. The common observation noted after polishing of seeds was the reduction in the level of carbohydrates and antioxidant potential, though the extent of reduction varied in the three cultivars. The only exception was GB, where there was no alteration in NO scavenging potential even after polishing. Our study showed the better performance of SW polished seeds with respect to higher amylose content and majority of the tested parameters governing antioxidant capacity and radical scavenging potential, thus highlighting the greater dietary significance of SW over the other two cultivars.     

Molecular identification of GHS-R and GPR38 in Suncus murinus

We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus.     

Spermidine and abscisic acid-mediated phosphorylation of a cytoplasmic protein from rice root in response to salinity stress

Importance of higher polyamines, spermidine, and spermine, in relation to the mechanism and adaptation to combat abiotic stress has been well established in cereals. Owing to their polycationic nature at physiological pH, polyamines bind strongly to negative charges in cellular components such as nucleic acids, various proteins, and phospholipids. To study the physiological role of polyamine during salinity stress, phosphorylation study was carried out in cytosolic soluble protein fraction isolated from the roots of salt tolerant (Nonabokra) and salt sensitive (M-1-48) rice cultivars treated with none (control), NaCl (150 mM, 16 h), spermidine (1 mM, 16 h) or with abscisic acid (100 μM, 16 h). A calcium independent auto regulatory 42 kDa protein kinase was found to phosphorylate myelin basic protein and casein but not histone. Interestingly, this was the only protein to be phosphorylated in root cytosolic fraction during NaCl/abscisic acid/spermidine treatment indicating its importance in salinity mediated signal transduction. This is the first report of polyamine as well as abscisic acid induced protein kinase activity in rice root in response to salinity stress.