Overproduction of soluble, extracellular cytotoxin α-sarcin inEscherichia coli

Parente, D.; Raucci, Giuseppe; D'alatri, L.; D'estais, G.; Novelli, Silvana; Pacilli, A.; Saccinto, M. P.; Mele, Antonio; Santis, Rita De

doi:10.1007/bf02760812

Cited by 5 publications

(4 citation statements)

References 23 publications

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“…Overproduction of active and extracellular cytotoxin, a-sarcin, was achieved when fused with protein A and a signal peptide sequence, with extracellular concentration of the recombinant protein reached up to 100 mg/l (Parente et al 1998). Protein A fusion was also successfully used for extracellular secretion of mouse-metallothinein for wastewater bioremediation (Cols et al 2001).…”

Section: Fusion Protein With Unknown Translocation Mechanismsmentioning

confidence: 99%

Extracellular recombinant protein production from Escherichia coli

Chen

2009

Biotechnol Lett

122

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Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

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Section: Fusion Protein With Unknown Translocation Mechanismsmentioning

confidence: 99%

Extracellular recombinant protein production from Escherichia coli

Chen

2009

Biotechnol Lett

122

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“…The production of recombinant proteins in the culture medium of E. coli cells has been reported by several groups [11–14], however, it might be rather regarded as an exceptional case with special proteins. In this report we first showed that a polysaccharide‐modifying enzyme, chitin deacetylase, was successfully produced in the culture medium of E. coli .…”

Section: Discussionmentioning

confidence: 99%

“…Herein we report the production of the recombinant chitin deacetylase in the culture medium of E. coli cells, with the aid of a signal sequence from Streptomyces lividans . The production of recombinant proteins in culture media of E. coli cells is rare [10], and it has been exclusively studied for the production of antibodies [11,12], a cytotoxin [13], and an insulin‐like growth factor binding protein [14]. The recombinant chitin deacetylases produced in the culture media of E. coli had comparable kinetic parameters to those of the original enzyme from the fungus, indicating that the expression system can be applied for further characterization studies of the enzymes, and the system is quite desirable for efficient recovery of the recombinant chitin deacetylase because no disruption steps of the cells are required.…”

Section: Introductionmentioning

confidence: 99%

Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells

et al. 1999

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With the aid of a signal sequence of a chitinase from Streptomyces lividans, a recombinant chitin deacetylase, whose gene originated from a Deuteromycete, Colletotrichum lindemuthianum, was produced in the culture medium of Escherichia coli cells, existing as a highly active form without the signal peptide. During the production of the recombinant chitin deacetylase, both a slight increase in the value of OD 600 nm in the culture medium and a drastic decrease in viable cell number were observed. When penta-N-acetyl-chitopentaose was used as the substrate, the recombinant chitin deacetylase had comparable kinetic parameters to those of the original enzyme from the fungus. The addition of a C-terminal six histidine sequence to the recombinant enzyme caused a slight decrease in the k cat value, and the further addition of a 12 amino acid sequence at its N-terminus caused a further decrease in the value. This production system allowed us to easily produce in the culture media the recombinant chitin deacetylases possessing as good properties as the original enzyme, without any disruption steps of the E. coli cells.z 1999 Federation of European Biochemical Societies.

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“…The proteins produced either accumulate intracellularly or are secreted into the periplasmic space. Some examples of protein secretion in the medium have also been reported (Parente et al 1998 ;Mergulhão et al 2005 ;Zhang et al 2006 ;Ni and Chen 2009 ;Schwarz et al 2012 ). The tendency for protein aggregation in the form of inclusion bodies, however, necessitates employing cumbersome protein purifi cation and refolding methods to obtain active protein (Lilie et al 1998 ;Sørensen and Mortensen 2005 ).…”

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confidence: 96%