TY - JOUR
T1 - Maximizing the recovery of the native p28 bacterial peptide with improved activity and maintained solubility and stability in Escherichia coli BL21 (DE3)
AU - Abuei, Haniyeh
AU - Pirouzfar, Mohammad
AU - Mojiri, Anahita
AU - Behzad-Behbahani, Abbas
AU - Kalantari, Tahereh
AU - Bemani, Peyman
AU - Farhadi, Ali
N1 - Funding Information:
This study was supported by a grant from Shiraz University of Medical Sciences , Shiraz, Iran under the agreement no. 22930 .
Publisher Copyright:
© 2022 Elsevier B.V.
PY - 2022/9
Y1 - 2022/9
N2 - p28 is a natural bacterial product, which recently has attracted much attention as an efficient cell penetrating peptide (CPP) and a promising anticancer agent. Considering the interesting biological qualities of p28, maximizing its expression appears to be a prominent priority. The optimization of such bioprocesses might be facilitated by utilizing statistical approaches such as Design of Experiment (DoE). In this study, we aimed to maximize the expression of “biologically active” p28 in Escherichia coli BL21 (DE3) host by harnessing statistical tools and experimental methods. Using Minitab, Plackett-Burman and Box-Behnken Response Surface Methodology (RSM) designs were generated to optimize the conditions for the expression of p28. Each condition was experimentally investigated by assessing the biological activity of the purified p28 in the MCF-7 breast cancer cell line. Seven independent variables were investigated, and three of them including ethanol concentration, OD600 of the culture at the time of induction, and the post-induction temperature were demonstrated to significantly affect the p28 expression in E. coli. The cytotoxicity, penetration efficiency, and total process time were measured as dependent variables. The optimized expression conditions were validated experimentally, and the final products were investigated in terms of expression yield, solubility, and stability in vitro. Following the optimization, an 8-fold increase of the concentration of p28 expression was observed. In this study, we suggest an optimized combination of effective factors to produce soluble p28 in the E. coli host, a protocol that results in the production of a significantly high amount of the biologically active peptide with retained solubility and stability.
AB - p28 is a natural bacterial product, which recently has attracted much attention as an efficient cell penetrating peptide (CPP) and a promising anticancer agent. Considering the interesting biological qualities of p28, maximizing its expression appears to be a prominent priority. The optimization of such bioprocesses might be facilitated by utilizing statistical approaches such as Design of Experiment (DoE). In this study, we aimed to maximize the expression of “biologically active” p28 in Escherichia coli BL21 (DE3) host by harnessing statistical tools and experimental methods. Using Minitab, Plackett-Burman and Box-Behnken Response Surface Methodology (RSM) designs were generated to optimize the conditions for the expression of p28. Each condition was experimentally investigated by assessing the biological activity of the purified p28 in the MCF-7 breast cancer cell line. Seven independent variables were investigated, and three of them including ethanol concentration, OD600 of the culture at the time of induction, and the post-induction temperature were demonstrated to significantly affect the p28 expression in E. coli. The cytotoxicity, penetration efficiency, and total process time were measured as dependent variables. The optimized expression conditions were validated experimentally, and the final products were investigated in terms of expression yield, solubility, and stability in vitro. Following the optimization, an 8-fold increase of the concentration of p28 expression was observed. In this study, we suggest an optimized combination of effective factors to produce soluble p28 in the E. coli host, a protocol that results in the production of a significantly high amount of the biologically active peptide with retained solubility and stability.
KW - CPP
KW - DoE
KW - E. coli BL21 (DE3)
KW - MCF-7
KW - Optimization
KW - p28
KW - Peptides/metabolism
KW - Solubility
KW - Escherichia coli/genetics
KW - Recombinant Proteins/metabolism
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U2 - 10.1016/j.mimet.2022.106560
DO - 10.1016/j.mimet.2022.106560
M3 - Article
C2 - 36031157
AN - SCOPUS:85136640261
SN - 0167-7012
VL - 200
SP - 106560
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
M1 - 106560
ER -