Expression and production of recombinant scorpine as a potassium channel blocker protein in Escherichia coli

Abstract

Scorpine is a cationic protein from the venom of Pandinus imperator, belonging to potassium channel blocker family, which has been shown to have antibacterial, antiviral, and antiplasmodia activities. In the present study, a pET-44a vector containing scorpine synthetic gene with T7 Promoter (pET 44a-His6-Nus-His6-tev-scorpine) was transferred into Escherichia coli Rosetta-gami B (DE3) for soluble expression of the protein in the cytoplasm and its overproduction. After confirming recombinant scorpine peptide expression using SDS-PAGE and Western blot, augmentation of production was performed during two stages. At first, effects of three parameters including carbon source concentration of medium, temperature, and induction time were investigated in terrific broth (TB) medium. Afterward, the overexpression was performed by response surface methodology in TB + glucose. Under the optimized conditions, the highest production of 3.5 g/L in the TB + glucose medium (7.5 g/L glucose, induction at OD 600 = 3.5 and 25 • C) was increased to 4.1 g/L in TB medium (2.5 g/L glycerol, induction at OD 600 = 0.7 and 25 • C). Then, in order to increase the amount of protein production, effects of carbon concentration in the fermenter under the primary optimized condition was investigated. The amount of produced recombinant protein increased from 0.12 to 2.1 g/L.H. The results were similar to previous studies on optimizing and increasing the production of recombinant protein and in particular recombinant scorpine. C 2018

Overexpression, overproduction, purification, and characterization of rhGH in Escherichia coli

“…Hence, the IPTG concentration of the medium can help improve the expression level of the recombinant protein [27]. Therefore, based on the conducted studies [15,23,29], the effect of IPTG concentration on rhGH production was investigated at two levels, 0.1 and 0.5 mM.…”

“…After identifying more effective factors, their optimization for increasing recombinant protein production is done by other statistical methods, especially by response surface and Taguchi methodologies. Response surface approach can evaluate the relationship between independent variables and effective response in tests designed to predict with more experiments in comparison with the Taguchi method [23]. In turn, the Taguchi approach can investigate more factors and qualitative parameters.…”

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

“…To extract soluble active proteins from inclusion bodies, the insoluble inclusion bodies must be first solubilized in denaturants ( Figure 5 ) and subsequently accompanied by a cycle of refolding (herein, referred to as the “one-step denaturing and refolding” procedure for comparison) [ 148 ]. This technique has been used for more than 20 years and is highly suitable for many bodily proteins, with approximately 40% being replenished into soluble and biologically active forms [ 138 , 149 ].…”

Revisiting the scope and applications of food enzymes from extremophiles

“…Escherichia coli remains the most popular expression platform for the production of recombinant proteins because of its numerous advantages. These include fast growth kinetics, high cell density, readily available and inexpensive growth media, and fast and easy DNA transformation (Levine, Gregorio, Jewett, Watts, & Oza, 2019; Menacho‐Melgar et al., 2020; Rosano & Ceccarelli, 2014; Rosano, Morales, & Ceccarelli, 2019; Seyfi, Babaeipour, Mofid, & Kahaki, 2019). A number of host yeast strains for recombinant production of proteins have also been reported, including Yarrowia lipolytica , Saccharomyces cerevisiae, Arxula adeninivorans, Pichia pastoris , Hansenula polymorpha, Debaryomyces hansenii, Schwanniomyces occidentalis, and Yarrowia lipolytica (Bischoff et al., 2019).…”