Materials and methods br Results

Materials and methods
Results
Discussion Since LF is a protease, rapid and direct purification is critical for production of functional rLF. Although previous investigators improved the rLF quality by simplifying purification steps, avoiding formation of inclusion body, decreasing degradation of rLF, and obtaining a high yield [19], [20], some disadvantages existed such as tedious changes of buffer in the process of purification, dialysis needed to remove the unbeneficial components in elution buffer, or additional steps necessary to cleave the tag from rLF. In this study, we successfully cloned the LF gene into pGEX-6P-1 and produced the tag (GST) free rLF by combining purification of the fusion protein with cleavage of GST. Above all, the whole process used only PBS buffer and dialysis was not required. Both the analysis of SDS–PAGE and Western blot assay showed that the purified protein was stable and did not experience apparent degradation. To obtain a high yield of rLF, BL21-codonPlus (DE3)-RIL was chosen as the expression strain. The strain contains extra copies of the argU, ileY, and leuW tRNA genes and is most suitable for the expression of genes with high AT content such as the LF gene [24]. In our small scale experiment, the yield of the purified rLF was 5mg/L of culture, similar to or even higher than the levels previously reported [19], [20]. Theoretically, an ideal method for purification must ensure that the purified protein remains biological activity. In the current study, both the cytotoxicity assay and immunological assay demonstrated that the purified rLF was highly functional [19]. The dose needed to lyse 50% of RAW264.7 nhs chemical when combined with rPA was as low as 2.7ng/ml. Meanwhile, Western blot assay and ELISA showed that the rLF maintained its immunogenicity. All the evidence indicated that this purification method can be used to efficiently produce the active rLF. Interestingly, we have also obtained an inactive mutant form of rLF, rLFm-Y236F. Although it was previously demonstrated that the residue 236 in LF was critical for LF toxicity, it is the first report to show that the mutation Y236F resulted in the loss of cytotixicity to RAW264.7 cells [25]. This mutant might be of significance in the study of anthrax pathogenesis and treatment.
Acknowledgments
Introduction Chronic fatigue syndrome (CFS) is a clinical condition characterized by long-lived, disabling fatigue associated with deficits in short-term memory, impairments in concentration, sleep disturbances, and skeletal muscle pain. The etiology of the disease is still unknown, but several studies suggest that an active viral infection and an immunological disorder may play fundamental roles. Onset of CFS after an acute or subacute viral infection, associated with mild fever, sore throat, muscular weakness, headache, migratory arthralgia, and other “flu-like” symptoms, is typical [1]. The prevalence of CFS ranges from seven to 267 cases per 10,000. The disease is more frequent in young adults and in white females [2]. Although some researchers consider CFS a psychological rather than a physical illness [3], several alterations support an organic origin of this disease. Many, although not all, CFS patients have atypical circulating lymphocytes and immune complexes [4], and increased concentrations of C-reactive protein and β-2 microglobulin [5], which suggests a chronic low-level activation of the immune system. This might be the response to a subclinical viral infection, since antibodies to Coxsackie B virus and Epstein Barr virus are commonly observed in these patients [6], [7]. Psychological factors have an important role in CSF: two-thirds of CFS patients suffer from a major depressive illness. These findings do not exclude the possibility of an organic origin of this disease, since depression is also characterized by specific neurotransmitter alterations. The most striking symptoms of the syndrome are muscle weakness and pain. In CFS patients, a deficiency of serum acylcarnitine has been found [8], which could induce abnormalities in mitochondrial activity. Reduced oxidative metabolism [9], [10] and higher plasma lactate concentration [11] have been reported. An impairment of mitochondrial oxidative phosphorylation in CFS is supported by the observation that in a group of patients suffering from this disease there is marked increase of intracellular pH after moderate exercise and a lower rate of ATP synthesis during recovery [12]. These findings may explain the muscle wasting due to an increased catabolic pathway observed in CFS.