New vectors for high level expression of recombinant proteins in bacteria

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein–protein or protein–membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

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Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassette

Journal of Virology ◽

10.1128/jvi.72.3.2289-2296.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 2289-2296 ◽

Cited By ~ 84

Author(s):

Bernard Massie ◽

France Couture ◽

Linda Lamoureux ◽

Dick D. Mosser ◽

Claire Guilbault ◽

...

Keyword(s):

Cell Lines ◽

Recombinant Proteins ◽

Recombinant Adenovirus ◽

Adenovirus Vector ◽

Expression Cassette ◽

Cell Protein ◽

Animal Cells ◽

High Level Expression ◽

Simplex Virus ◽

High Level

ABSTRACT We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(Δ2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce ΔR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5ΔR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.

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