increase in protein and plasmid yields of E. coli with optimized concentration of ampicillin as selection marker

Background: Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid

Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized

Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG [0.5 mM] and incubation with 0, 100, 200 and 300 micro g.mL[-1] ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve

Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 micro g.mL[-1] ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07x10[9], 3.21x10[9], 2.32x10[10] , 8.11x10[8], respectively. The plasmid yields were 55 ng.micro L[-1], 69 ng.micro L[-1], 164 ng.micro L[-1] and 41 ng.micro L[-1], respectively

Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration [200 micro g.mL[-1]] was significantly [p < 0.01] higher than other doses

Real-time PCR: an appropriate approach to confirm ssDNA generation from PCR product in SELEX process

Background: Aptamers are single stranded DNA [ssDNA] or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment [SELEX] method is much dependent on the successful conversion of double stranded DNA [dsDNA] to ssDNA

Objective: There are different methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased method, the method is not applicable in the initial rounds of SELEX due to more than 1015 different sequences. This study was designed to evaluate the efficiency of another technique for confirming the ssDNA generation in comparison to the polyacrylamide electrophoresis [PAGE] analysis

Materials and Methods: Real-time PCR was employed in the present study for PCR amplification of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis

Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a significant reduction of Tm from 73.8 to 41.5 [degree]C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was efficient enough to confirm ssDNA generation in accordance with the increasing the number of SELEX rounds

Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation

Altered Th17/Treg ratio in recurrent miscarriage after treatment with paternal lymphocytes and vitamin D3: a double-blind placebo-controlled study

Background: Recurrent miscarriage [RM] affects 2-5% of pregnant women. Paternal lymphocyte immunotherapy is a common treatment for RM patients but the outcome has not been consistent. Therefore, combined therapy with other immunosuppressive drugs such as 1a, 25-dihydroxy-vitamin-D3 [vitamin D3] may improve the outcome

Objectives: To investigate the effect of vitamin D3 on the balance of two essential T cells subsets, T helper [Th] 17 and T regulatory [Treg] cells, which regulate tolerance

Methods: The expression levels of CD4 and forkhead box protein 3 [FOXP3] in Treg cells, and the expression levels of CD4 and IL-17 in Th17 cells, were evaluated pre- and 3 months post-immunotherapy in RM patients treated with a combination of paternal lymphocytes and vitamin D3 compared with RM patients receiving lymphocyte immunotherapy alone

Results: Vitamin D3 therapy decreased the frequency of Th17 cells in addition to reducing the Th17/Treg ratio in peripheral blood of RM patients compared with the control group [p<0.05]

Conclusion: Considering that RM patients have a higher Th17/Treg ratio in peripheral blood, vitamin D3 may be a candidate therapeutic approach in this disease