BackgroundMild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies.ResultsHere we presented an efficient method for mild solubilization of inclusion bodies by using a freeze-thawing process in the presence of low concentration of urea. We used two different proteins to demonstrate the advantage of this method over the traditional urea-denatured method: enhanced green fluorescent protein (EGFP) and the catalytic domain of human macrophage metalloelastase (MMP-12_CAT). Firstly, PBS buffer at pH 8 containing different molar concentration of urea (0-8 M) were used to solubilize EGFP and MMP-12-CAT inclusion bodies and the solubility achieved in 2 M urea in PBS buffer by freeze-thawing method was comparable to that of PBS buffer containing 8 M urea by traditional urea-denatured method. Secondly, different solvents were used to solubilize EGFP and MMP-12_CAT from inclusion bodies and the results indicated that a wide range of buffers containing 2 M urea could efficiently solubilize EGFP and MMP-12_CAT inclusion bodies by freeze-thawing method. Thirdly, the effect of pH and freezing temperature on the solubility of EGFP and MMP-12_CAT inclusion bodies were studied, revealing that solubilization of inclusion bodies by freeze-thawing method is pH dependent and the optimal freezing temperature indicated here is −20°C. Forth, the solubilized EGFP and MMP-12_CAT from inclusion bodies were refolded by rapid dilution and dialysis, respectively. The results showed that the refolded efficiency is much higher (more than twice) from freeze-thawing method than the traditional urea-denatured method. The freeze-thawing method containing 2 M urea also effectively solubilized a number of proteins as inclusion bodies in E.coli.ConclusionsMild solubilization of inclusion body proteins using the freeze-thawing method is simple, highly efficient and generally applicable. The method can be utilized to prepare large quantities of bioactive soluble proteins from inclusion bodies for basic research and industrial purpose.

Recently, increasing attention is being paid to the viral etiology of atherosclerosis (AS). Human cytomegalovirus (HCMV) is thought to be the most possible etiological factor of AS. In our study, artery vascular tissue specimens derived from 75 patients with AS were studied for detection of HCMV immediately early (IE) and later (L) gene fragments by polymerase chain reaction (PCR) and in situ hybridization; sera collected from the same patients were examined for HCMV specific IgG and IgM by enzyme-linked immunosorbent assay (ELISA). Twenty two normal arterial tissues and sera were used as controls. We found that the positive rate of HCMV L and IE gene fragments were significantly higher in AS patients than those in controls. HCMV DNA were mainly observed in nucleus of endothelial cells and muscularis under intima as well as smooth muscle of media of AS area, rarely found in controls. These results suggested that HCMV infection may relate to the pathogenesis of AS; and the artery itself may be the site of HCMV latency. In addition, higher levels of HCMV IgG and IgM were found to be associated with virus persistence, indicating that a periodically active latent infection or a continuously active infection is presented in AS patients.

Specific virus-receptor interactions are important determinants in viral host range, tropism and pathogenesis, influencing the location and initiation of primary infection as well as viral spread to other target organs/tissues in the postviremic phase. Coxsackieviruses of Group B (CVB) and its six serotypes (CVB1-6) specifically interact with two receptor proteins, coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF), and cause various lesions in most permissive tissues. However, our previous data and other studies revealed that virus receptor-negative cells or tissues can be infected with CVB type 3 (CVB3), which can also effectively replicate. To study this interesting finding, we explored the possibility that exosomes are involved in CVB3 tropism and that exosomes functionally enhance CVB3 transmission. We found that exosomes carried and delivered CVB3 virions, resulting in efficient infection in receptor-negative host cells. We also found that delivery of CVB3 virions attached to exosomes depended on the virus receptor CAR. Importantly, exosomes carrying CVB3 virions exhibited greater infection efficiency than free virions because they accessed various entry routes, overcoming restrictions to viral tropism. In vivo experiments demonstrated that inhibition of exosome coupling with virions attenuated CVB3-induced immunological system dysfunction and reduced mortality. Our study describes a new mechanism in which exosomes contribute to viral tropism, spread, and pathogenesis.

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