IPTG-inducible promoter is popularly used for the expression of recombinant proteins. However, it is not suitable at the industrial scale due to the high cost and toxicity on the producing cells. Recently, a Self-Inducible Expression (SILEX) system has developed to bypass such problems using Hsp70 as an autoinducer. Herein, the effect of other heat shock proteins on the autoinduction of green fluorescent protein (EGFP), romiplostim, and interleukin-2 was investigated. For quantitative measurements, EGFP expression was monitored after double-transformation of pET28a-EGFP and pET21a-(Hsp27/Hsp40/Hsp70) plasmids into E.coli using fluorimetry. Moreover, the expression level, bacterial growth curve, and plasmid and expression stability were compared to an IPTG- inducible system using EGFP. Statistical analysis revealed a significant difference in EGFP expression between autoinducible and IPTG-inducible systems. The expression level was higher in Hsp27 system than Hsp70/Hsp40 systems. However, the highest amount of expression was observed for the inducible system. IPTG-inducible and Hsp70 systems showed more lag-time in the bacterial growth curve than Hsp27/Hsp40 systems. A relatively stable EGFP expression was observed in SILEX systems after several freeze–thaw cycles within 90 days, while, IPTG-inducible system showed a decreasing trend compared to the newly transformed bacteria. Moreover, the inducible system showed more variation in the EGFP expression among different clones than clones obtained by SILEX systems. All designed SILEX systems successfully self-induced the expression of protein models. In conclusion, Hsp27 system could be considered as a suitable autoinducible system for protein expression due to less metabolic burden, lower variation in the expression level, suitable plasmid and expression stability, and a higher expression level.
The residue substitutions identified might critically contribute as hot spots to different kinetic parameters of SKGs and might assist in further elucidation of structure/function relations and rational design of SKs with improved (fibrin-dependent) therapeutic properties.
Medical intervention with fibrinolytic drugs such as tissue plasminogen activator (tPA) and streptokinase (SK) is the principal treatment for life-treating thromboembolic disorders. Contrary to tPA, SK is a heterogenic and non-human (bacterial) protein produced by streptococci and its medical application may elicit sever immune and anaphylactic responses that restrict its utilization. Besides, human plasminogen (HPG) activation by SK is not blood-clot specific and associated with a risk of hemorrhage. Despite these limitations, comparative clinical trials on various thrombolytic agents suggested that SK is the most cost-effective fibrinolytic drug and almost as safe as its other counterparts such as tPA. Therefore, a number of studies were conducted to provide structurally modified SK with reduced immunogenicity, higher blood-clot specificity and half-lives. Although there are extensive overlaps in SK structural domains responsible for functionality, immunogenicity and stability that may limit its modifications, various strategies such as genetic manipulations (amino acid substitution /addition /deletion or domain fusions through production of chimeric SK proteins linked to HPG or hirudin) and chemical modification such as (homogenous/site-specific) PEGylation have been employed to develop a superior SK. In addition, data of the latest studies on SK screened from different streptococcal sources indicated the possibility of retrieving naturally occurring SKs with higher activities, less antigenicity and/or more fibrinspecificity. In the present review, after a survey on structure function relationships of SK domains and different strategies for SK improvement, recent advances and potential application of computer and matrix-based analyses for design and introduction of superior SKs will be presented.
Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasmin (Pm), is a protein secreted by groups A, C, and G streptococci (GAS, GCS, and GGS, respectively), with high sequence divergence and functional heterogeneity. While roles of some residual changes in altered SK functionality are shown, the underlying structural mechanisms are less known. Herein, using computational approaches, we analyzed the conformational basis for the increased activity of SK from a GGS (SKG132) isolate with four natural residual substitutions (Ile33Phe, Arg45Gln, Asn228Lys, Phe287Ile) compared to the standard GCS (SKC). Using the crystal structure of SK.Pm catalytic complex as main template SKC.μPm catalytic complex was modeled through homology modeling process and validated by several online validation servers. Subsequently, SKG132.μPm structure was constructed by altering the corresponding residual substitutions. Results of three independent MD simulations showed increased RMSF values for SKG132.μPm, indicating the enhanced structural flexibility compared to SKC.μPm, specially in 170 and 250 loops and three regions: R1 (149-161), R2 (182-215) and R3 (224-229). In parallel, the average number of Hydrogen bonds in 170 loop, R2 and R3 (especially for Asn228Lys) of SKG132 compared to that of the SKC was decreased. Accordingly, residue interaction networks (RINs) analyses indicated that Asn228Lys might induce more level of structural flexibility by generation of free Lys256, while Phe287Ile and Ile33Phe enhanced the stabilization of the SKG132.μPm catalytic complex. These results denoted the potential role of the optimal dynamic state and stabilized catalytic complex for increased PA potencies of SK as a thrombolytic drug.
Background and Objectives: Cholera disease remains an important global health problem affecting 3-5 million subjects worldwide. Outer membrane vesicles (OMVs) have been found in a variety of Gram-negative bacteria and act as protective transport vesicles. The aim of this study was to evaluate Immune responses against Vibrio cholerae O1 El Tor clinical strain OMV and compare it with killed whole cell (KWC), complex of (KWC-OMV) as well as the internationally licensed oral cholera vaccine, Dukoral, in serum and intestinal secretions of mice. Materials and Methods: OMVs were prepared by using modified detergent-centrifugation procedure from V. cholerae O1 El Tor clinical strain from 2005 outbreak. The ultrastructure and content of OMVs were investigated via the Scanning Elec- tron Microscopy (SEM) and SDS-PAGE analysis. Three doses of oral immunization were adjusted and total IgG and IgA in serum and intestinal secretion were measured by enzyme-linked immunosorbent assay (ELISA). Results: Extracted OMVs from the V. cholerae were spherical vesicles with a size ranging from 10 to 300 nm. OMV-im- munized mice showed an increased level of total IgG and IgA both in serum and intestinal secretion when compared to the negative controls. Also, there existed a higher level of secretory IgA than the total IgG, suggesting the most of protection against V. cholerae colonization provided by sIgA. Conclusion: Our findings revealed that oral immunization with V. cholerae OMVs might induce a long-term immunity, es- pecially when administered in combination with KWC. This study tested the adjuvant activity of OMVs and may be useful in future nano vaccine research.
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