Heterologous Expression and Metal-Binding Characterization of a Type 1 Metallothionein Isoform (OsMTI-1b) from Rice (Oryza sativa)

Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni²⁺, Cd²⁺, Zn²⁺ and Cu²⁺ ions was analyzed. The results demonstrated that this isoform has ability to bind Ni²⁺, Cd²⁺ and Zn²⁺ ions in vitro, whereas it has no substantial ability to bind Cu²⁺ ions. From competitive reaction with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni²⁺/Cd²⁺ > Zn²⁺ > Cu²⁺     

Inherent anti-HIV activity of biocompatible anionic citrate-PEG-citrate dendrimer

Molecular Biology Reports - The development of new combinations to empower better protection against HIV infection is particularly important. Anionic polymers can block HIV infection. In the...     

Purification of Antibacterial CHAP<Subscript>K</Subscript> Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Therapeutic LysK-CHAP is a potent anti-staphylococcal protein that could be utilized as an antibiotic substitute. Intein-mediated protein purification is a reasonable and cost-effective method that is most recently used for recombinant therapeutic protein production. Intein (INT) is the internal parts of the protein that can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. INT sequence and their characteristic of self-cleavage by thiol induction, temperature, and pH changes are used for protein purification. The current study presents the expression of CHAP_K262 domain of LysK gene that is fused with INT/chitin-binding sequence while evaluating its purification procedure and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The coding gene sequence of LysK-CHAP (CHAP_K262) in pET22-b was amplified with polymerase chain reaction (PCR); the digested product was then cloned into the pTXB1 vector. Electrophoresis confirmed the cloning accuracy of the gene. The pTXB1-CHAP_K262 plasmid was transformed to the Escherichia coli ER₂₅₆₆ (E. coli ER₂₅₆₆) expression strain and analyzed for expression of the recombinant protein by SDS-PAGE and Western blotting methods. Finally, CHAP_K262 was purified by chitin affinity column using INT tag technology and confirmed by SDS-PAGE. Lytic activity of the purified protein was investigated by disk diffusion method. Cloning of CHAP_K262 into the pTXB1 vector, which comprised INT/chitin-binding sequence, was successfully achieved. The SDS-PAGE data also revealed successful expression of the CHAP_K262-INT fusion protein and Western blotting method validated the accuracy of the protein. Moreover, purification of CHAP_K262 protein was induced by dithiothreitol (DTT) and confirmed by SDS-PAGE. Finally, inhibition zone in MRAS culture medium confirmed antibacterial activity of the protein. Application of intein-mediated antibacterial protein is an appropriate and streamlined method for one-step purification of CHAP_K262 as a therapeutic and antibacterial protein. Self-cleaving tags like intein are cost-effective and could be used as a proper purification method for industrial purposes.     

Compensatory Role of Insulin in the Extinction but Not Reinstatement of Morphine-Induced Conditioned Place Preference in the Streptozotocin-Induced Diabetic Rats

Neurochemical Research - Insulin receptors are distributed in the whole brain, including different parts of the reward circuit that modulate dopamine as the primary neurotransmitter implicated in...     

Development of a method for the quantitative evaluation of microorganism sensitivity to antibiotics utilizing disks. The determination of the minimal doxycycline concentration that depresses microorganism growth using disks containing this antibiotic]

A quantitative method for estimation of microbial sensitivity to doxycycline with the use of discs containing 10 gamma of the antibiotic was developed. The antibiotic concentrations in the agar were determined at a distance equal to the radius of the growth inhibition zone with the help of a curve of the dependence of the logarithm of the doxycycline concentration in agar at the period of the average critical time of the zone formation equal to 5 hours and the distance from the disc center. The antibiotic concentration in the agar at the zone edges was almost the same as the MIC of doxycycline against the test-cultures determined with the method of serial dilutions in agar.     

Determination of rifamycin B activity in culture liquids and in preparations with varying degrees of purity]

A possibility of using the biological method of rifamycin B activity determination in the fermentation broth and dry preparations of various purity levels was studied. It was found that the biological method was useful only for determination of rifamycin B activity in preparations containing not less than 850 gamma/mg of the main product. When the activity of rifamycin B was determined in the fermentation broth and crude preparations containing less than 800 gamma/mg of the main product, the results of the biological assay were always higher as compared to those of spectrophotometrical estimation. It was accounted for the effect of other rifamycin types possessing high biological activity.