Synthesis, purification and biological properties of a truncated mutant form of human tissue plasminogen activator produced in E. Coli

Fromage, Nadine; Denèfle, Patrice; Cambou, Bernard; Duchesne, Marc; Joyeux, Christian; S, Kovarik; Marin, J.; Imbault, F.; Uzan, A.; Cartwright, Terence

doi:10.1016/0268-9499(91)90022-v

Cited by 5 publications

(3 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To obtain have to be removed, and the polypeptide changed into a buffer under oxidizing conditions. The most frequently used way to reduce the concentration of denaturant and reducing reagents is to dilute the polypeptide directly is also possible to dialyse the protein against different buffers' 1,30,35,74,76,89,99 or to remove the chemicals by gel filtration. 86 Starting the refolding by dilution changes the solvent properties surrounding the denatured protein instantly.…”

Section: Renaturation and Reoxidation Of Solubilized Polypeptidesmentioning

confidence: 99%

Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies

Fischer¹,

Sumner²,

Goodenough³

1993

Biotech & Bioengineering

182

116

View full text Add to dashboard Cite

Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies, In case of expression of eukaryotic proteins containing cysteine, which may form disulfide bonds in the native active protein, often nonnative inter- and intramolecular disulfide bonds exist in the inclusion bodies. Hence, several methods have been developed to isolate recombinant eukaryotic polypeptides from inclusion bodies, and to generate native disulfide bonds, to get active proteins. This article summarizes the different steps and methods of isolation and renaturation of eukaryotic proteins containing disulfide bonds, which have been expressed in E. coli as inclusion bodies, and shows which methods originally developed for studying the folding mechanism of naturally occurring proteins have been successfully adapted for reactivation of recombinant eukaryotic proteins.

show abstract

Section: Renaturation and Reoxidation Of Solubilized Polypeptidesmentioning

confidence: 99%

Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies

Fischer¹,

Sumner²,

Goodenough³

1993

Biotech & Bioengineering

182

116

View full text Add to dashboard Cite

show abstract

“…Recent advances in recombinant DNA technology have significantly facilitated the production of many recombinant proteins of therapeutic importance (Furman et al, 1987;Fromage et al, 1991). Among the many potential host cell systems used for the production of recombinant proteins, Escherichia coli is the most attractive expression system because of its ease of growth and of genetic manipulation (Georgiou, 1988).…”

Section: Introductionmentioning

confidence: 99%

Enhanced protein renaturation by temperature-responsive polymers

Lin¹,

Lin²,

Chiu³

et al. 2000

Biotechnol. Bioeng.

View full text Add to dashboard Cite

The application of temperature‐sensitive polymer (PNIPAAm) for the renaturation of β‐lactamase from inclusion bodies was investigated. It was observed that PNIPAAm was more effective than PEG in enhancing protein renaturation. At a concentration of 0.1%, PNIPAAm improved the yield of β‐lactamase activity by 41% from 46.5 to 65.4 IU/mL, compared to 26% with PEG from 46.5 to 58.7 IU/mL. Kinetic study indicated that PNIPAAm did not significantly affect the initial rate of protein renaturation but did increase final activity yield. In the presence of PEG and PNIPAAm, the activity yields increased with temperature, indicating that hydrophobic interactions between denatured protein and polymer molecules contributed to the enhanced protein renaturation with polymers. The sequential addition approach, aiming at enhancing protein renaturation by reducing local protein concentration during renaturation, was also shown effective in enhancing protein renaturation, especially in the presence of polymers. With the sequential addition approach, the activity yield was increased by 60.5% from 46.5 to 74.6 IU/mL with PNIPAAm. Similar behavior was also observed with PEG. PNIPAAm exhibited similar behavior as PEG on the renaturation of β‐lactamase in terms of temperature effect and concentration effect, indicating that the mechanism for enhanced protein renaturation for the two polymers might be similar. PNIPAAm exhibits a lower critical solution temperature (LCST) of 32°C and can be effectively separated from aqueous solution and recycled. A protein renaturation process employing PNIPAAm, which offers the advantages of enhanced renaturation efficiency, minimum loss of protein aggregates, and ease of polymers recycling, was proposed. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 505–512, 2000.

show abstract

“…45 Several attempts to produce ht-PA or a truncated form in E. coli have been reported. 29,32,[45][46][47][48] Recombinant t-PA (rt-PA) accumulated as inclusion bodies in the cytoplasm. t-PA best exemplifies the challenges associated with the production of multidisulfide complex proteins in E. coli by refolding from inclusion bodies.…”

Section: Case Studies Of the Production Of Therapeutic Proteins Example 1: T-pamentioning

confidence: 99%

Refolding of therapeutic proteins produced inEscherichia coli as inclusion bodies

1999

View full text Add to dashboard Cite

Overexpression of cloned or synthetic genes in Escherichia coli often results in the formation of insoluble protein inclusion bodies. Within the last decade, specific methods and strategies have been developed for preparing active recombinant proteins from these inclusion bodies. Usually, the inclusion bodies can be separated easily from other cell components by centrifugation, solubilized by denaturants such as guanidine hydrochloride (Gdn‐HCl) or urea, and then renatured through a refolding process such as dilution or dialysis. Recent improvements in renaturation procedures have included the inhibition of aggregation during refolding by application of low molecular weight additives and matrix‐bound renaturation. These methods have made it possible to obtain high yields of biologically active proteins by taking into account process parameters such as protein concentration, redox conditions, temperature, pH, and ionic strength. © 1999 John Wiley & Sons, Inc. Biopoly 51: 297–307, 1999

show abstract

Synthesis, purification and biological properties of a truncated mutant form of human tissue plasminogen activator produced in E. Coli

Cited by 5 publications

References 10 publications

Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies

Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies

Enhanced protein renaturation by temperature-responsive polymers

Refolding of therapeutic proteins produced inEscherichia coli as inclusion bodies

Contact Info

Product

Resources

About