Iranian Journal of Biotechnology
Volume:9 Issue: 4, Autumn 2011

The mature core protein of the Hepatitis C virus (HCVC173) carrying pelB as a signal peptide (PelB::core) was overexpressed in Escherichia coli as 18% and 23.3% of the host’s total protein, in flask and fermentor cultivation, respectively. A final specific yield of 25 ± 1 mg HCVC173/g dry cell weight and an overall productivity of 51±1 mg HCVC173/l/h were obtained in the stirred-tank fermentor. The recombinant PelB::core protein was overexpressed as the inclusion body (IB) form, higher than the expected level when compared to the HCVC173, which was also showed by the analysis of secondary structure of mRNAs and calculation of the Codon Adaptation Index of the gene. The results showed that the combined effects of protein fusion and the signal sequence significantly enhanced the production of recombinant mature HCVC173 in E. coli. Therefore, the fusion form of the mature HCV core protein and the conditions defined in this study provide an alternative strategy for HCVC173 production in high cell density culture of E. coli.

Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro refolding to increase recombinant proteins production. Additives increase protein refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process. In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA) in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and Dithiothreitol (DTT) on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In refolding step, L-arginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM. Glutathione pairs (GSH-GssG) showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the presence of higher concentration of L-arginine (> 500 mM) or pH (>10) is not associated with higher activity.

Linear programming problems with multiple optimal solutions are challenging due to the choice of multiple strategies resulting in the same optimal value of the objective function. However, searching for these solutions is a tedious task especially when using mixed integer linear programming (MILP), as previously applied to metabolic models. Therefore, judgment on plurality of optimal metabolic flux distributions (solutions) a priori to applying MILP approach could prevent unnecessary computations. In this work for the first time, the reduced cost coefficients for the non-basic variables in a current solution of a metabolic model were utilized to inspect the possibility of multiple optimal flux distributions. If there exists at least one non-basic variable with zero reduced cost coefficient, multiplicity of optimal solution may occur where MILP can be used to find these optimal solutions. This approach was implemented on a metabolic network of Bacillus subtilis aiming to reduce the cell energy requirement. Solving the model at fixed specific growth rate of 0.4 h-1 resulted in minimum energy requirement of 12.67 mmol (g h)-1. Inspection of reduced cost coefficients showed that six non-basic variables had zero reduced cost coefficients at current optimal solution, which proves that there can exist multiple optimal solutions. Subsequently by applying MILP, five optimal flux distributions at minimized energy requirement were identified, among which one showing no acid production and minimum glucose consumption rate was selected as the superior solution.

Alkaline proteases are of industrial importance, mainly in the detergent industry. In this study, the extracellular alkaline serine protease gene, aprE, from Bacillus clausii was amplified by PCR and further cloned into B. subtilis WB600 using the pWB980 expression vector. Measurement of protease activity in the recombinant B. subtilis WB600 harboring the pWB980-aprE showed a yield of 1020 enzyme units ml-1, approximately 3 folds higher than the native B. clausii. Characterization of the alkaline protease by SDS-PAGE and zymogram analyses indicated a molecular weight of 31 kDa for the cloned enzyme with biological activity. DNA sequence analysis and the deduced amino acid sequence revealed 98% homology with the extracellular alkaline serine protease from Bacillus clausii KSM-K16.

Single Nucleotide Polymorphisms (SNPs) are the most usual form of polymorphism in human genome. Analyses of genetic variations have revealed that individual genomes share common SNP-haplotypes. Particular pattern of these common variations forms a block-like structure on human genome. In this work, we develop a new method based on the Perfect Phylogeny Model to identify haplotype blocks using samples of individual genomes. We give a rigorous definition of the quality of the partitioning of haplotypes into blocks and describe a greedy algorithm for finding the proper partitioning in case of perfect and semi-perfect phylogeny. It is shown that the minimum number of tagSNPs in a haplotype block of Perfect Phylogeny can be obtained by a polynomial time algorithm. We apply the algorithm to haplotype data of human chromosome 21 and compare the results with other previously developed methods through simulations. The results demonstrate that our algorithm outperforms the conventional implementation of the Four Gamete Test approach which is the only available method for haplotype block partitioning based on Perfect Phylogeny.

In this study, immobilization of partially purified almond (Amygdalus communis) β-galactosidase on Con A layered calcium alginate-cellulose beads has been investigated. Immobilized β-galactosidase retained 72% of the initial activity after crosslinking by glutaraldehyde. Both soluble and immobilized enzyme exhibited the same pH and temperature optima at pH 5.5 and 50 oC, respectively. However, the immobilized enzyme showed a remarkable broadening in pH and temperature-activity profiles as compared to the native enzyme. Immobilized enzyme was significantly more stable against thermal denaturation at 60 oC. Immobilized β-galactosidase exhibited 67% residual activity in the presence of 5% D-galactose while its soluble counterpart retained only 35% activity under identical conditions. Soluble enzyme showed 69% residual activity after exposure to pepsin (0.15 mg/mL) for 1 h whereas the immobilized β-galactosidase was more stable and retained nearly 84% activity under identical experimental conditions. The activity of immobilized enzyme was enhanced to 156% whereas soluble β-galactosidase showed an enhancement upto 134% when exposed to trypsin (0.1 mg/mL) for 1 h. Moreover, immobilized β-galactosidase exhibited greater enhancement in enzyme activity against exposure to various ions present in milk such as Na+, K+, Ca+2, Mg+2 and citrate ions. The higher concentration of lactose was hydrolyzed from whey as compared to the hydrolysis from milk by immobilized enzyme at 50 °C and 60 °C in batch process. Lactose was hydrolyzed to 86% and 78%, respectively after 20 d continuous operation of reactors at the flow rates of 20 mL/h and 30 mL/h, respectively.

Endophytic actinomycetes were isolated from surface sterilized leaves of Catharanthes roseus (L.) G.Don of family Apocynaceae. A total of 38 endophytic actinomycetes were recovered on Starch Casein Agar. Among the 38 isolates 20 morphologically different isolates were screened for antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris and antifungal activity against fungi Candida albicans, Botrytis cinerea, Curvularia lunata, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani. 65% of the isolates exhibited antimicrobial activity. Among the 20 isolates tested two isolates Cr-12, Cr-20 exhibited highest activity against the test organisms.

Modified Cetyltrimethylammonium bromide (CTAB) protocol for DNA isolation was developed from leaf tissues of Phyllanthus emblica for obtaining high quality genomic DNA. Fresh leaves of three different maturity were analyzed for yield and quality of DNA. Acidity was determined in three different maturity of leaves viz. tender, intermediate and mature and their influence on DNA quality was determined. Drastic reduction of pH was the primary cause for poor quality of DNA. However, high quality DNA isolation was achieved by stabilizing the pH by addition of NaOH during different stages of DNA isolation process. The present protocol yielding high quality intact DNA for genetic fingerprinting as well as for amplification of chloroplast genes for molecular analysis.