Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus is a recognized enteropathogen causing diarrhea in humans and is one of the major causes of seafoodborne gastroenteritis. An important virulence factor is thermostable direct hemolysin (TDH), a pore-forming toxin, which is able to lyse eukaryotic cells. The active toxin is a tetramer of four identical protein subunits, which is secreted by the pathogen after cleavage of a signal peptide. To establish diagnostic detection systems for TDH we expressed the hemolysin with and without the signal peptide in a prokaryotic cell-free system to obtain pure toxin. In order to purify and to facilitate the isolation from cell lysates we synthesized TDH variants with different tags. Important regulatory sequences for cell-free protein synthesis as well as sequences for N-terminal Strep-tag and C-terminal 6xHis-tag were added by a two-step PCR. For the expression in the cell-free system these linear tdh templates were subjected directly to prokaryotic cell extracts. Protein yields were in the range of 500-600 μg/ml for the preproteins and approx. 300-400 μg/ml for the mature proteins. The identities of expressed proteins were further confirmed by SDS-PAGE, immunological and MALDI-TOF mass spectrometric analyses. The functionality of newly synthesized toxin variants was tested by performing qualitative and semiquantitative hemolysis assays. Cell-free produced mature TDH and its variants were active while the preprotein and its derivatives lacked hemolytic activity. A C-terminal 6xHis-tag showed less influence on functionality compared to the N-terminal Strep-tag.

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

“…Over the last decade, cell-free methods have proven themselves as a valuable platform allowing the synthesis of many different protein classes including membrane proteins [1], [2], [3], [4], [5], [6], [7], proteins with posttranslational modifications [8], [9], [10], [11], [12], [13], [14] and even toxic proteins [15], [16], [17]. Many problematic issues connected with a cell-based expression of proteins, such as protein insolubility and toxicity, can be circumvented by the use of tailor-made cell-free expression systems.…”

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

“…This system is widely used for synthesis of MPs in the presence of membrane solubilization supplements in the form of nanodiscs, detergents, proteoliposomes etc. added externally into the cell-free reaction [5,10]. Nanodiscs are synthetic discoidal nanoparticles consisting of a phospholipid bilayer surrounded by two copies of membrane scaffold proteins (MSPs).…”

“…This eukaryotic cell-free system offers additional advantages in the form of native, ER-derived endogenous microsomes. Such microsomes contain the entire translocon machinery responsible for proper folding of MPs [8][9][10][11][12][13]. Recently, the potassium channel KcsA was synthesized successfully in this system [13].…”

Cell-Free Synthesis Meets Antibody Production: A Review

“…Nowadays, the system has proven its potential to serve as a reliable, versatile and highly flexible protein production platform, enabling the synthesis of difficult-to-express proteins, as for example membrane proteins [7] and even toxic proteins [6,44]. Cell-free protein synthesis has been shown to be scalable from the microliter to liter scale [4].…”