Dataset Information

Simple piggyBac transposon-based mammalian cell expression system for inducible protein production.

ABSTRACT: Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.

SUBMITTER: Li Z

PROVIDER: S-EPMC3612669 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Simple piggyBac transposon-based mammalian cell expression system for inducible protein production.

Li Zhijie Z Michael Iacovos P IP Zhou Dongxia D Nagy Andras A Rini James M JM

Proceedings of the National Academy of Sciences of the United States of America 20130308 13

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continu ...[more]

PMID: 23476064

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Project description:BACKGROUND: After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction) prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. RESULT: The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS) system.The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. CONCLUSION: Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene independently from external inducers or drastic changes in metabolic conditions and enabling tight regulation of expression.

Dataset Information

Simple piggyBac transposon-based mammalian cell expression system for inducible protein production.

Publications

Simple piggyBac transposon-based mammalian cell expression system for inducible protein production.

Similar Datasets

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