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BMC Biotechnology (1)
Biotechnology Letters (1)
Bohlmann, Holger (2)
Shah, Kausar Hussain (2)
Abbas, Amjad (1)
Ali, Muhammad Amjad (1)
Plattner, Stephan (1)
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author:("Shah, causar Hussain")
Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.
Electronic supplementary material
The online version of this article (doi:10.1007/s10529-013-1180-z) contains supplementary material, which is available to authorized users.
Fusion protein; His-tag; Maltose-binding protein; Tobacco etch virus; Thionin; Thioredoxin
pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage
Ali, Muhammad Amjad
The production of transgenic plants, either for the overproduction of the protein of interest, for promoter: reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming.
We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1: DsRed marker and a CaMV:: GUS cassette within the T-DNA and is useful for the production of promoter: GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1: DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia.
The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest.
Transient expression; pPZP family vectors; Marker gene; Agroinfiltration; DsRed; Agrobacterium; Arabidopsis transformation
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