Expression and Purification of Plant-derived Enzymes
Lehtinen, Antti (2020)
Lehtinen, Antti
2020
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Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:amk-2020052213082
https://urn.fi/URN:NBN:fi:amk-2020052213082
Tiivistelmä
Gerbera hybrida is a plant in the asteraceae family that has been proposed as a new model species for development biology studies. Other well-known model species include the fruit fly and Escherichia coli, for example. Of flowering plants there is only one well known model species so far, Arabidobsis thaliana.
One feature of interest is Gerberas secondary metabolite pathways, notably gerberin, parasorboside. Gerberin and parasorboside are known to function in the defense mechanism of Gerbera against pathogens and herbivores.
The aim of this work was to express and purify a set of enzymes of Gerbera hybrida in Escherichia coli strain Lemo 21. These enzymes are known or are proposed to function in the biosynthesis of gerberin and parasorboside. These enzymes are G2PS1, G2PS2, PKS8 (gerbera polyketides, ~45 kDa), GTKPR1, GRED1 and GRED2 (gerbera reductases, ~35 kDa). These enzymes are still not very well known, and they have not been used in research anywhere else.
The heterologous exspression was begun by culturing the expression host constructs that had been provided. The hosts were previously transformed with pHis8, a pET-28 vector that included the gene necessary for the expression of the enzymes in the multicloning site. The expressed proteins had an 8x histidine tag to the N-terminus which was used to purify and identify the product. The host cells were lysated and the expressed enzymes purified with His SpinTrap kit. The fractions of the purification steps were collected and analyzed with Bradford assay. The fractions were additionally analyzed with SDS-PAGE and dot blot methods to determine the product size and the presence of the 8x histidine tag respectively.
It was discovered that GRED1 and GRED2 did not produce as strong a signal in the dot blot analysis as the other enzymes. This might have been due to incorrect folding of the protein where the histidine tag was folded inside the protein structure and thus was not purified in the correct fraction and did not produce a strong signal in dot blot. The expression and purification methods were modified and the yield of GRED1 was increased by 51.45 %. All the expressed enzymes were stored for further study.
One feature of interest is Gerberas secondary metabolite pathways, notably gerberin, parasorboside. Gerberin and parasorboside are known to function in the defense mechanism of Gerbera against pathogens and herbivores.
The aim of this work was to express and purify a set of enzymes of Gerbera hybrida in Escherichia coli strain Lemo 21. These enzymes are known or are proposed to function in the biosynthesis of gerberin and parasorboside. These enzymes are G2PS1, G2PS2, PKS8 (gerbera polyketides, ~45 kDa), GTKPR1, GRED1 and GRED2 (gerbera reductases, ~35 kDa). These enzymes are still not very well known, and they have not been used in research anywhere else.
The heterologous exspression was begun by culturing the expression host constructs that had been provided. The hosts were previously transformed with pHis8, a pET-28 vector that included the gene necessary for the expression of the enzymes in the multicloning site. The expressed proteins had an 8x histidine tag to the N-terminus which was used to purify and identify the product. The host cells were lysated and the expressed enzymes purified with His SpinTrap kit. The fractions of the purification steps were collected and analyzed with Bradford assay. The fractions were additionally analyzed with SDS-PAGE and dot blot methods to determine the product size and the presence of the 8x histidine tag respectively.
It was discovered that GRED1 and GRED2 did not produce as strong a signal in the dot blot analysis as the other enzymes. This might have been due to incorrect folding of the protein where the histidine tag was folded inside the protein structure and thus was not purified in the correct fraction and did not produce a strong signal in dot blot. The expression and purification methods were modified and the yield of GRED1 was increased by 51.45 %. All the expressed enzymes were stored for further study.