Title:Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia
coli at High Productivity Using SUMO Fusion and an Adjusted
Expression Strategy
Volume: 29
Issue: 4
Author(s): Shirin Tarahomjoo*, Mojgan Bandehpour, Mohammad Aghaebrahimian and Salimeh Ahangaran
Affiliation:
- Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO),
Karaj 31975/148, Iran
Keywords:
CRM197, diphtheria toxin, recombinant, solubility, SUMO protease, SUMO tag.
Abstract:
Background: CRM197, a non-toxic diphtheria toxin variant, is widely used as a
polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197
commercial production is limited due to the low yield of Corynebacterium diphtheriae C7
(197) tox-. Developing an efficient method for recombinant CRM197 production reduces production
costs and is critical for expanding the application coverage of related medical products and basic research.
Escherichia coli is a frequently used host for heterologous protein synthesis. However, the
primary limitation of this system is the inclusion body formation and the low yield of active protein
recovery.
Objective: As a result, we attempted to produce CRM197 in the soluble form in E. coli using a
small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein
production.
Methods: CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused
to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197
in E. coli, a response surface method (RSM) experimental design was used based on three
factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium.
Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed
using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA
column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197
and CRM197.
Results: When HSCRM197 was expressed in E. coli under common expression conditions (37ºC,
1000 μM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when
the RSM-derived expression conditions were used (30ºC, 510 μM inducer, and 200 mM sorbitol),
the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity
was 32.67 μg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However,
the activity of CRM197 was greater than that of HSCRM197.
Conclusion: These findings established the utility of the method developed in this study to produce
CRM197 for medical applications.