Fractional factorial approach combining 4 Escherichia coli strains, 3 culture media, 3 expression temperatures and 5 N-terminal fusion tags for screening the soluble expression of recombinant proteins

2012 ◽  
Vol 84 (2) ◽  
pp. 204-213 ◽  
Author(s):  
Christophe Noguère ◽  
Anna M. Larsson ◽  
Jean-Christophe Guyot ◽  
Christophe Bignon
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Culture Media ◽  
Soluble Expression ◽  
Fractional Factorial ◽  
Factorial Approach

scholarly journals Critical points worthy of consideration in the soluble expression of recombinant proteins in Escherichia coli: the accuracy of the solubility prediction tools versus experimental results

2020 ◽  
Author(s):  
Fatemeh Malaei ◽  
mohammad javad rasaee
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Soluble Expression ◽  
Experimental Results ◽  
High Yield ◽  
P Value ◽  
Gene Design ◽  
E Coli ◽  
Prediction Tools ◽  
First Choice

Abstract Purpose: Recombinant proteins have become increasingly important items in research and industry. Due to its low cost, high yield and rapid growth rate, Escherichia coli (E. coli) is the first choice as host for the production of recombinant proteins. The expression of recombinant proteins in E. coli systems often result in inclusion bodies lacking proper folding and structure. In silico bioinformatics prediction tools may be promising in optimal expression of soluble recombinant proteins. Materials and methods: In this review, we aimed at making critical recommendations on how to improve the soluble expression of recombinant proteins. Furthermore, we compared the solubility of recombinant proteins using bioinformatics prediction tools versus experimental results. Data were analyzed using SPSS software. Results: Some recommendations worthy of consideration in gene design and expression were reminded. The results of a comparison between bioinformatics and experimental methods revealed that no significant coordination existed. RPSP and SOLpro showed higher sensitivity (43.5% and 56.5%, respectively) and specificity (52.9% and 47.1%, respectively), when compared to FoldIndex and PSoL. The results from p-value and roc curve indicated the effect of MW, helix percentage and aliphatic index on protein solubility (p-value< 0.05). Conclusions: This review discusses efficient expression of soluble recombinant proteins. The bioinformatics prediction tools were examined for their sensitivity and specificity. MW, helix percentage and aliphatic parameters should be considered in gene design.

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

scholarly journals The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum

2008 ◽  
Vol 74 (23) ◽  
pp. 7431-7433 ◽  
Author(s):  
Mónica Martínez-Alonso ◽  
Nuria González-Montalbán ◽  
Elena García-Fruitós ◽  
Antonio Villaverde
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Recombinant Proteins ◽  
Fluorescent Protein ◽  
Native Structure ◽  
Functional Quality ◽  
Soluble Aggregates ◽  
Recombinant Polypeptides ◽  
Green Fluorescent

ABSTRACT We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure.

scholarly journals Production of recombinant proteins from Plasmodium falciparum in Escherichia coli

Biomédica ◽  
2016 ◽  
Vol 36 ◽  
Author(s):  
Ángela Patricia Guerra ◽  
Eliana Patricia Calvo ◽  
Moisés Wasserman ◽  
Jacqueline Chaparro-Olaya
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
Recombinant Proteins

<p><strong>Introducción.</strong> La producción de proteínas recombinantes es fundamental para el estudio funcional de proteínas de <em>Plasmodium</em> <em>falciparum</em>. Sin embargo, las proteínas recombinantes de <em>P</em>. <em>falciparum</em> están entre las más difíciles de expresar y cuando lo hacen usualmente se agregan dentro de cuerpos de inclusión insolubles.</p><p><strong>Objetivo.</strong> Evaluar la producción de cuatro proteínas de <em>P. falciparum</em>, usando como sistema de expresión dos cepas de <em>Escherichia coli </em>genéticamente modificadas para favorecer la producción de proteínas heterólogas y establecer una reserva de proteínas recombinantes puras y solubles y producir anticuerpos policlonales a partir de ellas.<strong></strong></p><p><strong>Materiales y métodos.</strong> Las proteínas recombinantes, las cuales correspondían a secuencias parciales de PfMyoA (Miosina-A) y PfGAP50 (proteína-asociada a glideosoma-50 kDa) y a las secuencias completas de PfMTIP (proteína de interacción con Miosina-A) y PfGAP45 (proteína asociada a glideosoma-45 kDa), fueron expresadas como proteínas de fusión con GST y luego purificadas y usadas para producir anticuerpos policlonales en ratón.</p><p><strong>Resultados.</strong> La expresión de las proteínas recombinantes fue mucho más eficiente en la cepa BL21-CodonPlus (la cual expresa tRNAs escasos en las bacterias silvestres), que en la cepa BL21-pG-KJE8. En contraste, aunque la cepa BL21-pG-KJE sobreexpresa chaperonas, no redujo la formación de cuerpos de inclusión. <strong>Conclusión.</strong> El uso de cepas de <em>E</em>. <em>coli</em> genéticamente modificadas fue fundamental para alcanzar altos niveles de expresión de las cuatro proteínas recombinantes evaluadas y permitió obtener dos de ellas en forma soluble. La estrategia utilizada permitió expresar cuatro proteínas recombinantes de <em>P</em>. <em>falciparum</em> en cantidad suficiente para inmunizar ratones y producir anticuerpos policlonales, y además conservar proteína pura y soluble de dos de ellas, para ensayos futuros.</p>

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