Optimization of culture media for extracellular expression of streptokinase in Escherichia coli using response surface methodology in combination with Plackett-Burman Design

Mojtaba Aghaeepoor,Mohammad Reza Akbari Eidgahi,Ali Akbarzadeh,Ehsan Dehnavi,Maryam Ahmadi,Aliakbar Shabani,Farzad Kobarfard

doi:10.4314/tjpr.v16i11.1

Abstract

Purpose: To investigate the enhancement of streptokinase extracellular expression in Escherichia coli by adjusting culture media.Methods: Screening of 10 chemical factors (EDTA, peptone, glycine, triton X-100, glycerol, K2HPO4, KH2PO4, Ca2+ (calcium chloride), yeast and NaCl) in order to increase the secretion of extracellular protein was carried out by response surface methodology (RSM). The method was also employed to optimize the concentrations of critical factors that had been determined in the screening step.Results: The results indicate that glycine, triton X-100 and Ca2+ were the most effective chemical factors in terms of increase in extracellular expression of streptokinase with optimum levels of 0.878, 0.479 and 0.222 %, respectively. Expression of streptokinase under optimum concentrations of critical permeabilizing factors led to a 7-fold increase in the quantity of secreted recombinant protein (5824 U/mL) compared to the initial level (802 U/mL).Conclusion: The results show that medium optimization using RSM is effective in improving extracellular streptokinase expression. The optimization medium is considered fundamental and useful for efficient production of streptokinase on a large scale.Keywords: Streptokinase, Response surface methodology, Membrane permeabilization, Extracellular secretion

Highlights

Streptokinase (SK) is a single chain (47 kDa) protein that has received much attention as a commonly used thrombolytic agent for treating cardiovascular disease such as heart attack or stroke [1]
Heterologous expression in E. coli could bring about numerous advantages, accumulation of the expressed protein in the cytosol as a biologically inactive protein aggregate is a significant problem that could be overcame by extracellular expression of recombinant protein [5,6]
The existence certain values of triton X-100 and glycine in the culture medium had a strong effect on extracellular production and could greatly increase both inner and outer cell membrane permeability [12]

Summary

Introduction

Streptokinase (SK) is a single chain (47 kDa) protein that has received much attention as a commonly used thrombolytic agent for treating cardiovascular disease such as heart attack or stroke [1]. Medium optimization is one of the most commonly applied approaches to facilitate translocation through membranes Some medium supplements such as lysozyme, magnesium, calcium, EDTA, glycine, and triton X-100 could increase membrane permeability and subsequent protein secretion to the culture medium [9,10,11]. The existence certain values of triton X-100 and glycine in the culture medium had a strong effect on extracellular production and could greatly increase both inner and outer cell membrane permeability [12]. Divalent cations, such as Ca+2 and Mg+2 in medium rich with glycine can effectively protect cells from lysing [13]. An RSM designs the least number of experiments to achieve these goals

Objectives

Results

Conclusion