Optimization of rPDT fusion protein expression by Escherichia coli in pilot scale fermentation: a statistical experimental design approach

Nasser Nassiri Koopaei,Hamid Moloudian,Parissa Khadiv-Parsi,Mohammad Ali Mazlomi,Abbas Kebriaeezadeh,Mohammad Reza Khoshayand,Mahdi Aminian,Roya Solhi

doi:10.1186/s13568-018-0667-3

Nasser Nassiri Koopaei, Hamid Moloudian + Show 6 more

Open Access

https://doi.org/10.1186/s13568-018-0667-3

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Abstract

High yield recombinant protein production is highly desirable for biotechnological purposes. In the design of recombinant expression conditions, a number of essential central elements such as expression strain, type of medium, bioprocess optimization, and mathematical modeling should be considered. Well-designed industrial scale production of one recombinant protein with optimized influential parameters and yield can address the cost and production reproducibility issues. In the present study, statistical experimental design methodology was used to investigate the effect of fermentation conditions (dissolved oxygen, IPTG, and temperature) on rPDT production by Escherichia coli. rPDT is a recombinant fusion protein consisting of three different protein domains including the N-terminal 179 amino acid fragment of the S1 subunit of pertussis toxin, the full-length genetically detoxified diphtheria toxin (CRM197), and the 50 kDa tetanus toxin fragment C. A 15 Box–Behnken design augmented with center points revealed that IPTG and DO at the center point and low temperature will result in high yield. The optimal condition for rPDT production were found to be 100 µM IPTG, DO 30% and temperature 20 °C.

Highlights

Recombinant DNA technology has made it possible to produce different recombinant proteins in amounts required for research, clinical and industrial purposes (Rosano and Ceccarelli 2014; Pavlou and Reichert 2004; Kebriaeezadeh et al 2013)
The bacterial strain expressing rPDT protein was selected and the rPDT production was optimized using statistical design. rPDT protein composes of the immunoprotective S1 fragment of pertussis toxin, the full-length nontoxic diphtheria toxin (CRM197), and fragment C of tetanus toxin (Aminian et al 2007; Eisel et al 1986). This fusion protein has been expressed in E. coli carrying the recombinant plasmid and has a molecular weight of 161 kDa that is recognizable by specific antibodies against the three toxins. rPDT expression is inducible by IPTG (Aminian et al 2007; Esposito and Chatterjee 2006; Soria-Guerra et al 2009). rPDT is a fusion protein containing the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin and fragment C of tetanus toxin which has been engineered to serve as a candidate for the vaccination against diphtheria–tetanus–pertussis (Aminian et al 2007)
Optimization of rPDT production by statistical experimental design An optimization approach was applied in the present study using the results from a prior screening study

Summary

Introduction

Recombinant DNA technology has made it possible to produce different recombinant proteins in amounts required for research, clinical and industrial purposes (Rosano and Ceccarelli 2014; Pavlou and Reichert 2004; Kebriaeezadeh et al 2013). Escherichia coli is known as a preferable expression system, because of its rapid growth and simplicity of cultivation. In spite of all the advantages, production of recombinant proteins involves very complicated steps that require application of sophisticated control and optimization approach (Andersen and Krummen 2002).

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