Expression optimization of anti-CD22 scFv-Apoptin fusion protein using experimental design methodology

Background: Design of experiments is a rapid and cost-effective approach for optimization of recombinant protein production process. In our previous study, we generated a potent dual-acting fusion protein, anti-CD22 scFv-apoptin, to target B-cell malignant cell lines. In the present investigation, we report the effect of different variables on the expression levels of this fusion protein

Methods: Four variables [cell optical density at induction, IPTG concentration, induction temperature, and induction time] were tested using experimental design

Results: Our findings demonstrated that among the examined variables, only the induction time had a significant positive effect on the protein expression yield

Conclusion: Experimental design was successfully applied in this study. The optimized condition obtained in the current study can be applied in future commercial production of this novel fusion protein

Design, synthesis and biological evaluation of 4-benzamidobenzoic acid hydrazide derivatives as novel soluble epoxide hydrolase inhibitors

Inhibitors of soluble epoxide hydrolase [sEH] represent one of the novel pharmaceutical approaches for treating hypertension, vascular inflammation, pain and other cardiovascular related diseases. Most of the potent sEH inhibitors reported in literature often suffer from poor solubility and bioavailability. Toward improving pharmacokinetic profile beside favorable potency, two series of 4-benzamidobenzoic acid hydrazide derivatives with hydrazide group as a novel secondary pharmacophore against sEH enzyme were developed. The designed compounds were synthesized in acceptable yield and their in vitro assay was determined. Most of the synthesized compounds have appropriate physical properties and exhibited considerable in-vitro sEH inhibitory activity in comparison with 12-[3-Adamantan-1-yl-ureido]-dodecanoicacid [AUDA], a potent urea-based sEH inhibitor. 4-[2-[4-[4-chlorobenzamido] benzoyl]hydrazinyl]-4-oxobutanoic acid 6c was found to be the most potent inhibitor with inhibitory activity of 72% targeting sEH enzyme

Optimum conditions of radioligand receptor binding assay of ligands of benzodiazepine Receptors

To obtain drugs which are more selective at benzodiazepine [BZD] receptors, design and synthesis of functionally selective ligands for BZD receptors is the current strategy of our pharmaceutical chemistry department. The affinity of newly synthesized ligands is assessed by radioligand receptor binding assays. Based on our previous studies, 2-phenyl-5-oxo-7-methyl-1,3, 4-oxadiazolo[a,2,3]-pyrimidine [compound A] was chosen for design and synthesis of new triazole derivatives as GABA[A] BZD receptor agonist. The cortical membrane of male Sprague-Dawley rats was prepared as the source of the BZD receptors. Different concentrations of membrane protein and [[3]H]-flumazenil were incubated at room temperature at different time periods to reach the steady-state. To saturate the receptors, increased amounts of radioligand were incubated with membrane protein. The bound and un-bound ligands were separated by centrifugation. The affinity of compound A was measured in competition studies at optimum conditions by displacement of [[3]H]-Flumazenil from rat cortical membrane. Based on results, the optimum conditions of radioligand receptor binding assay of benzodiazepines were 35 min incubation of ligands with 100 micro g cortical membrane protein and 8.6 x 10[-5] nmole [3]H-flumazenil in a final volume of 0.5 mL Tris-HCl buffer [50 mM, pH 7.4] at 30 [degree sign]C. The binding parameters of [[3]H]-flumazenil, B[max] and K[d] were determined through saturation studies as 0.638 +/- 0.099 pmol/mg and 1.35 +/- 0.316 nM respectively. The affinity of compound A was 1.9 nM comparable with diazepam [1.53nM]. This finding makes the compound an interesting lead for further optimization. Starting from this compound, new ligands were synthesized and screened in-vitro by competitive binding assays

Challenges to design and develop of DNA aptamers for protein targets. I. optimization of asymmetric PCR for generation of a single stranded DNA library

Aptamers, or single stranded oligonucleotides, are produced by systematic evolution of ligands by exponential enrichment, abbreviated as SELEX. In the amplification and regeneration step of SELEX technique, dsDNA is conversed to ssDNA. Asymmetric PCR is one of the methods used for the generation of ssDNA. The purpose of this study was to design a random DNA library for selection of aptamers with high affinity for protein targets and develop an efficient asymmetric PCR amplification. Thus, the influence of factors including annealing temperature, number of amplification cycles, primer ratio, Mg[2+] concentration and the presence of a PCR enhancer on the amplification of the desired product were evaluated. Results obtained by agarose gel electrophoresis showed that the annealing temperature of 64 [degree sign]C, Mg[2+] concentration of 0.25 mM, reverse to forward primer ratio of 15:1, amplification cycle of 25 and the presence L-ectoin as a PCR enhancer with the concentration of 0.4 M were the optimal conditions. Our results supported that the yield of this type of ssDNA production is sufficient for combinatorial screening of aptamers

Preparation of testosterone using microbial metabolite of cholesterol

Natural sterols are potential raw materials for the prepartion of pharmacologically active steroid products. This paper details preparation of testosterone, one of the steroidal hormones, through microbial metabolism of cholesterol followed by chemical reduction. The side chain of cholesterol was selectively cleaved by Mycobacterium smegmatis PTCC 1307, and then selective reduction of resulting 17-keton group of androst-4-ene-3, 17-dione [AD], the main product of microbial transformation, with sodium borohydride at -10°C gave testosterone in high yeild [90%]. Assignment of structures were made by IR, NMR, Mass, and UV spectroscopic data

Comparison of biodistribution of 111 I - tropolone leukocytes and 125 I - human nonspecific polyclonal IgG in normal and induced inflammation mice for detection of inflammation.

Human nonspecific polyclonal IgG and granulocytes, which accumulate in inflammation foci, were radiolabeled with 125I and "'In-Tropolone, respectively. Biodistribution of these two radiolabels was assessed in normal and inflammation-induced mice. 125I-IgG showed better localization to the inflammated areas. Blood levels with "'In-Tropolone leukocytes were lower at all time points. In addition, the inflammatory thigh-to-blood ratios showed an improvement, whereas the ratios of inflammatory thigh-to-other normal tissues were higher for 125I-IgG than "'In-Tropolone leukocytes. In conclusion, labeled IgG due to better localization in inflammated sites and higher target-to-background ratios is more suitable agent than labeled leukocytes for immunoscintigraphy of inflammation