Lab-scale Preparation of Recombinant Human Insulin-like Growth Factor-1 in Escherichia coli and its Potential Safety on Normal Human Lung Cell Line.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) is structurally similar to insulin and acts as an endocrine hormone secreted by the liver. OBJECTIVE: Production of recombinant human IGF-1 (rhIGF-1) in Escherichia coli (E.coli) and evaluation of its proliferation stimulatory activity. METHODS: hIGF-1 gene cloned into pBSK (+) simple vector was transformed into TOP 10 chemically competent cells of E. coli. Polymerase chain reaction (PCR) was achieved using specific hIGF-1 gene primers to confirm the successful transformation. To express the rhIGF-1 in E. coli (Rosetta (DE3) pLysS); the hIGF-1 gene was cloned into the pET-15b expression vector and then the recombinant pET-15b/IGF-1 vector was transformed into a chemically prepared competent expression bacterial cells; Rosetta (DE3) pLysS. The rhIGF-1 was expressed as insoluble aggregates called inclusion bodies (IBs) using a 2 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) inducer. IBs were solubilized in a denatured form using 6 M guanidinium hydrochloride (GdmCl), followed by in vitro protein refolding using the rapid dilution method. The refolded hIGF-1 was purified using the HiTrap- ANX anion exchange column. Western blot and ELISA using rabbit polyvalent anti-hIGF- 1 were performed to confirm the protein antigenic identity. Cell proliferation activity of rhIGF-1 was testified on normal human lung cell line (WI-38). RESULTS: rhIGF-1 was purified from the HiTrap-ANX column at a concentration of 300 µg/ml. Western blot showed a single 7.6 kDa band obtained in the induced Rosetta (DE3) pLYsS. ELISA confirmed the molecular identity of the rhIGF-1 epitope, the concentration of purified rhIGF-1 obtained from the ELISA standard curve using rhIGF-1 reference protein as a standard was 300 µg/ml, and activity on WI-38 cells was 2604.17I U/mg. CONCLUSION: Biologically active native rhIGF-1 protein was successfully expressed. Patents related to the preparation of IGF-1 were mentioned along the text.

Association of sex hormones with metabolic syndrome among Egyptian males.

BACKGROUND: Studies have shown that testosterone and estradiol (E2) are associated with metabolic syndrome (MetS). To our knowledge, few studies, if any about the association of endogenous sex hormones with MetS have been done in Egypt. AIM: To study the relation between endogenous sex hormones and MetS among Egyptian males. SUBJECTS AND METHOD: For the study, 80 Egyptian males were enrolled: 40 males with MetS and 40 healthy age-matched males. Anthropometric measurements and blood pressure were taken for both groups. FBG, TC, HDL-C, TG, testosterone, and E2 levels were determined; LDL-C was calculated. RESULTS: Males with MetS had significantly lower testosterone levels and significantly higher E2 levels compared to those without MetS (p value 0.0001). The lowest quartile of testosterone was most prevalent among males with MetS (19/40 males, 47.5%) compared to those without MetS (0/40 males, 0%, p value 0.011). Estradiol in the third quartile was most prevalent among males with MetS (19/40 males, 47.5%) compared to those without MetS (1/40 males, 2.5%, p value 0.0001). Serum testosterone and E2 levels were independent predictors of MetS with optimum cut off value (≤2.37ng/ml) for testosterone and (>16.78pg/ml) for E2. CONCLUSION: Endogenous testosterone and estradiol are independently associated with MetS with potential utility as predictors of MetS.