Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology

Abstract

Background:The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium.Materials and Methods:Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli (E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3), followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM). Finally, the best culture medium was selected.Results:Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml). Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media.Conclusion:In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

The expression and construction of engineering Escherichia coli producing humanized AluY RNAs

“…The reason of inducing AluY RNA yield decreased maybe that RNA easily degrade. Shafiee et al [ 29 ] found that the IPTG concentration had the most effect on protein expression and the optimal IPTG concentration was 1 mmol/l; in this paper, we found that optimal IPTG concentration was 0.1–0.4 mg/ml (0.42–1.68 mmol/l) (Fig. 1 d).…”

“…Shafiee et al [ 29 ] showed the highest level of DT386-BR2 fusion protein expression was attained after 2 h of incubation with IPTG and increasing the incubation time did not affect the amount of expression; in this paper, we found that AluY RNA yield reached peak at 4 h of IPTG induction, remarkably decreased after 8 h with IPTG induction. The reason of inducing AluY RNA yield decreased maybe that RNA easily degrade.…”

Caracterização de L-Asparaginase de <i>Erwinia chrysanthemi</i> melhorada por evolução sintética de proteí­nas e otimização das condições de produção

“…Assim como verificado em outros trabalhos (ASHAYERI-PANAH et al 2017;SHAFIEE et al 2017) a melhor expressão ocorreu a 37 °C.…”

“…A concentração do indutor também afeta a expressão da proteína. A baixa concentração do indutor pode resultar em uma indução ineficiente e por outro lado, os indutores adicionados em excesso podem resultar em efeitos tóxicos, incluindo crescimento celular reduzido e, finalmente, redução da concentração de proteína recombinante (RAMIREZ et al 1994;SHAFIEE et al 2017). Nesse trabalho foi possível verificar que a concentração do indutor afetou os valores obtidos de atividade específica, principalmente no caso de temperaturas mais baixas.…”

“…Ao analisar as condições 7, 8, 10 e 11, as quais todas elas foram submetidas à temperatura de indução de 37ºC, os valores de atividade específica obtidos não apresentaram diferença significativa, sendo o valor médio de 282,0 ± 10,9 U/mg, quase o dobro do maior valor de atividade encontrado nas condições 4 e 5 (148,0 ± 23,1 U/mg), e aproximadamente 3 vezes maior do que valor de atividade encontrado nas condições 1 e 2 (95,0 ± 3,9 U/mg). Nesse trabalho, a temperatura de indução de 37ºC apresentou os melhores resultados, assim como verificado em outros trabalhos (ASHAYERI-PANAH et al 2017;SHAFIEE et al 2017;SOLEYMAN et al 2016;MALIK et al 2016).…”