In vivo efficiency of the produced recombinant lysostaphin antimicrobial peptide in treatment of methicillin-resistant Staphylococcus aureus (MRSA) skin infection in a mouse model.

Background and Objectives: Staphylococcus simulans secretes an antimicrobial compound called lysostaphin, which has bactericidal properties. It destroys staphylococci through the hydrolysis of peptidoglycan in the cell wall. Therefore, this unique property indicates the high ability of lysostaphin in the treatment of staphylococcal infections and is considered as an anti-staphylococcal agent. Materials and Methods: Escherichia coli BL21 (DE3) competent cells were transformed with pET32a-lysostaphin clone and induced by isopropyl-ß-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant lysostaphin -A-based ointment was used for external wound healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: Our results showed the recombinant protein was produced exactly. The results of checkerboard tests showed MIC, MBC and antibacterial activity test an acute reduction of cell viability during the use of lysostaphin, and SEM results approved the intense wrecking effects of lysostaphin in combination on bacterial cells. Macroscopic findings and microscopic data showed that the recombinant lysostaphin ointment was effective on excisional wound healing. Conclusion: Our findings proved that the recombinant lysostaphin ointment was effective on wound healing due to Staphylococcus aureus infection.

Production of mutant streptavidin protein and investigation of its effect on the performance of streptavidin.

Objectives: Streptavidin is a versatile protein in cell science. The tetramer structure of streptavidin plays a key role in this binding, but this form interferes with some assays. If monomer streptavidin is still capable of binding to biotin, it can overcome the limitations of the streptavidin application. So, we examined the elimination of tryptophan 120 and its effect on the function of streptavidin. Materials and Methods: Mutant streptavidin gene was synthesized in a pBSK vector. Then it was ligated to the pET32α vector. This vector is expressed in Escherichia coli BL21 (DE3) pLysS host. After purification and refolding of the recombinant protein, its structure was analyzed on the SDS_PAGE gel. Recombinant streptavidin binding affinity to biotin was evaluated by spectrophotometric and HABA color compound. Results: Mutant streptavidin gene was successfully expressed in E. coli BL21 (DE3) pLysS host and the purified protein was observed as a single band in the 36 kDa area. The best condition for dialysis was PBS buffer+arginine. The molar ratio of biotin/protein of mutant streptavidin was not only near but also more than standard protein. Mutant streptavidin remained in the monomeric state in the presence or absence of biotin. Conclusion: Results of this study showed that 120 tryptophan is one of the most important factors in tetramer streptavidin formation and its deletion produces the monomer form that has a high binding affinity to biotin. This mutant form of streptavidin can therefore be used in studies requiring monovalent binding as well as in studies facing limitations due to the size of streptavidin tetramer.

Ib-AMP4 antimicrobial peptide as a treatment for skin and systematic infection of methicillin-resistant Staphylococcus aureus (MRSA).

Objectives: Antimicrobial peptide compounds (AMPs) play important roles in the immune system. They also exhibit significant anti-tumor and antibacterial properties. Most AMPs are cationic and are able to bind bacterial cell membranes through electrostatic affinity. Ib-AMP4 is a plant-derived AMP that exerts rapid bactericidal functions. In the present study, the antibacterial efficiency of the produced recombinant Ib-AMP4 in elimination of Methicillin-resistant Staphylococcus aureus (MRSA) bacterial infection, was investigated under in vitro and in vivo situations. Materials and Methods: The synthesized Escherichia coli codon-optimized gene sequences of the Ib-AMP4 were expressed in E. coli BL21 (DE3) pLysS. The recombinant Ib-AMP4 was purified and refolding conditions were optimized. The antibacterial efficiency of the refolded peptide against MRSA was tested under in vivo and in vitro situations for treatment of skin and systematic infection of MRSA in a mouse model. Results: Antibacterial assays confirmed the antibacterial function of Ib-AMP4 against MRSA. SEM results proved the destructive effects of applying Ib-AMP4 on MRSA biomembrane. Time-kill curve and growth kinetic assay illustrated rapid antibacterial activity of the produced Ib-AMP4. Moreover, Ib-AMP4 showed significant infection treatment ability in a mouse model and all infected mice receiving Ib-AMP4 protein survived and there was no trace of bacteria in their blood samples. Conclusion: The results confirmed the rapid antibacterial potential of the produced recombinant Ib-AMP4 to be used for efficient treatment of MRSA infection.

Cloning, expression and purification of antigenic fragments of the Ureaplasma urealyticum UreD protein and its value in serology.

Background and Objectives: The detection of Ureaplasma urealyticum is usually done through culture. With the change of the smallest effective factor in culture, we face the lack of growth of these bacteria, which is one of the important reasons to find a suitable alternative for the diagnosis of this bacterium. UreD is a protected gene in this bacterium. The aim of this was to evaluate the ability of antigenic regions of UreD protein to bind to patients' serum antibodies. Materials and Methods: Antigenic regions of UreD protein were predicted using IEDB software with five different methods: Emini Surface Accessibility Prediction, Kolaskar and Tongaonkar Antigenicity, Chou and Fasman beta turn prediction, Karplus and Schulz flexibility scale, Ellipro-Epitope prediction based upon structural protrusion. Antigenic regions of UreD gene was clonned, expressed and purified. The antigenicity of this recombinant protein against the antibodies in the serum of people infected with U. urealyticum infections was checked in western blotting. Results: The results showed that the antigenic regions of the UreD protein was producted and its antigenicity was demonstrated in western blotting. Moreover, all sera from patients infected with U. urealyticum reacted to the recombinant antigen. Conclusion: Specimens from people infected with U. urealyticum infection was positive in Western blotting suggesting that UreD protein has antigenic properties. Therefore, it can be used as a suitable candidate for the design of diagnostic kits and U. urealyticum vaccine.

Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity.

BACKGROUND: Streptavidin is a protein produced by Streptomyces avidinii with strong biotin-binding ability. The non-covalent, yet strong bond between these two molecules has made it a preferable option in biological detection systems. Due to its extensive use, considerable attention is focused on streptavidin production by recombinant methods. METHODS: In this study, streptavidin was expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS cells and purified by affinity chromatography. Various dialysis methods were employed to enable the protein to refold to its natural form and create a strong bond with biotin. RESULTS: Streptavidin was efficiently expressed in E. coli. Streptavidin attained its natural form during the dialysis phase and the refolded protein bound biotin. The addition of proline or arginine to the dialysis buffer resulted in a refolded streptavidin with greater affinity for biotin than refolding in dialysis buffer with no added amino acids. CONCLUSION: Dialysis of recombinant streptavidin in the presence of arginine or proline resulted in proper refolding of the protein. The recombinant dialyzed streptavidin bound biotin with affinity as great as that of a commercial streptavidin.

Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes.

BACKGROUND: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. OBJECTIVES: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activity of this protein was investigated. MATERIALS AND METHODS: The expression of hyaluronidase antigenic fragments was optimized using IPTG concentration, time of induction, temperature, culture, and absorbance of 0.6-0.8-1 at 600 nm. Afterwards, the expressed proteins were purified and the enzymatic activity was assessed by turbid metric method. RESULTS: Data indicated that maximum protein is produced in OD = 0.8, 0.5 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG), 37ºC, NB 1.5x, without glucose, incubated for overnight. The enzymatic activity of the recombinant protein was similar to the commercial form of hyaluronidase. CONCLUSIONS: The results showed that an antigenic fragment of the recombinant hyaluronidase protein from S. pyogenes has a considerable enzymatic activity. It can be suggested to use it for medical purposes. In addition, applications of bioinformatics software would facilitate the production of a smaller protein with same antigenic properties and enzymatic activity.