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Expression Profiling and Recombinant...

Mishler-Elmore, John W.

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Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase./
作者:	Mishler-Elmore, John W.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2020,
面頁冊數:	175 p.
附註:	Source: Dissertations Abstracts International, Volume: 82-03, Section: B.
Contained By:	Dissertations Abstracts International82-03B.
標題:	Biochemistry. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28107941
ISBN:	9798662597556

Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase.
Mishler-Elmore, John W.

Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase. - Ann Arbor : ProQuest Dissertations & Theses, 2020 - 175 p.

Source: Dissertations Abstracts International, Volume: 82-03, Section: B.

Thesis (Ph.D.)--Ohio University, 2020.

This item must not be sold to any third party vendors.

Extensin peroxidases play a critical role in plant cell growth and are believed to play equally important roles in defense from pathogenesis and mechanical stress. By catalyzing the covalent polymerization of extensin proteins, they participate in the formation of the cell plate for cell division and help to reinforce the wall-preventing pathogen infection. Due to it's anionic character and catalytic processivity, TomEP is a particularly unique extensin peroxidase that requires much less time and enzyme than other extensin peroxidases to crosslink extensin substrate. Previous work identified the TomEP gene, and established methods to produce functional enzyme through heterologous expression in E. coli. This work aimed to expand upon these previous efforts by characterizing TomEP expression, TomEP function in vivo, and design a purification scheme to produce milligram-level quantities of pure enzyme for crystallization. An expression profile of TomEP was compiled using both qPCR analysis and promoter-GUS fusion experiments to provide data describing normal expression and response to wounding. Basal TomEP expression was demonstrated to be significantly higher in roots than in flowers, stems, or leaves. Through the same methods, wounding treatments were shown to increase TomEP expression in tomato roots from one to four hours, followed by attenuation for the following sixteen hours.The foundations of gain and loss-of-function experiments were pursued in an attempt to discern TomEP's influence on di-isodityrosine and pulcherosine content in tomato cell walls, using overexpression and CRISPR knock-out strategies. Overexpression lines of tomato and Arabidopsis were generated using Agrobacterium mediated methods, though these efforts failed to produce verifiable protein product, despite expression being observed on the RNA level. Transient expression in tobacco epidermal cells was successful however, allowing for in vivo analysis of TomEP activity, though no clear link between TomEP and di-isodityrosine or pulcherosine formation could be made. CRISPR knock-out lines of tomato were successfully generated, with two lines likely possessing homozygous or chimeric mutant alleles and fifteen others appearing to have heterozygous mutant alleles. No cell wall analysis of mutants could be performed without further breeding and characterization, but the path has been blazed for future research. Utilizing an established oxido-shuffling strategy to fold protein extracted from E. coli inclusion bodies, milligram quantities of recombinant TomEP were produced and purified. The previous purification scheme was unable to accommodate larger reaction volumes and additional protein product and required alterations to scale up production. Techniques utilized include ammonium sulfate precipitation, immobilized metal affinity purification, anion exchange chromatography, and size exclusion chromatography. Using these methods, the milligram threshold was exceeded using the new purification scheme, enabling future x-ray crystallography.

ISBN: 9798662597556Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Plant

Expression Profiling and Recombinant Production of TomEP, a Tomato Extensin Peroxidase.
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300 $a 175 p.
500 $a Source: Dissertations Abstracts International, Volume: 82-03, Section: B.
500 $a Advisor: Held, Michael A., II.
502 $a Thesis (Ph.D.)--Ohio University, 2020.
506 $a This item must not be sold to any third party vendors.
520 $a Extensin peroxidases play a critical role in plant cell growth and are believed to play equally important roles in defense from pathogenesis and mechanical stress. By catalyzing the covalent polymerization of extensin proteins, they participate in the formation of the cell plate for cell division and help to reinforce the wall-preventing pathogen infection. Due to it's anionic character and catalytic processivity, TomEP is a particularly unique extensin peroxidase that requires much less time and enzyme than other extensin peroxidases to crosslink extensin substrate. Previous work identified the TomEP gene, and established methods to produce functional enzyme through heterologous expression in E. coli. This work aimed to expand upon these previous efforts by characterizing TomEP expression, TomEP function in vivo, and design a purification scheme to produce milligram-level quantities of pure enzyme for crystallization. An expression profile of TomEP was compiled using both qPCR analysis and promoter-GUS fusion experiments to provide data describing normal expression and response to wounding. Basal TomEP expression was demonstrated to be significantly higher in roots than in flowers, stems, or leaves. Through the same methods, wounding treatments were shown to increase TomEP expression in tomato roots from one to four hours, followed by attenuation for the following sixteen hours.The foundations of gain and loss-of-function experiments were pursued in an attempt to discern TomEP's influence on di-isodityrosine and pulcherosine content in tomato cell walls, using overexpression and CRISPR knock-out strategies. Overexpression lines of tomato and Arabidopsis were generated using Agrobacterium mediated methods, though these efforts failed to produce verifiable protein product, despite expression being observed on the RNA level. Transient expression in tobacco epidermal cells was successful however, allowing for in vivo analysis of TomEP activity, though no clear link between TomEP and di-isodityrosine or pulcherosine formation could be made. CRISPR knock-out lines of tomato were successfully generated, with two lines likely possessing homozygous or chimeric mutant alleles and fifteen others appearing to have heterozygous mutant alleles. No cell wall analysis of mutants could be performed without further breeding and characterization, but the path has been blazed for future research. Utilizing an established oxido-shuffling strategy to fold protein extracted from E. coli inclusion bodies, milligram quantities of recombinant TomEP were produced and purified. The previous purification scheme was unable to accommodate larger reaction volumes and additional protein product and required alterations to scale up production. Techniques utilized include ammonium sulfate precipitation, immobilized metal affinity purification, anion exchange chromatography, and size exclusion chromatography. Using these methods, the milligram threshold was exceeded using the new purification scheme, enabling future x-ray crystallography.
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650 4 $a Plant sciences. $3 3173832
650 4 $a Physiology. $3 518431
650 4 $a Botany. $3 516217
653 $a Plant
653 $a Cell Wall
653 $a Extensin
653 $a Extensin Peroxidase
653 $a Peroxidase
653 $a HRGP
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690 $a 0487
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710 2 $a Ohio University. $b Chemistry and Biochemistry (Arts and Sciences). $3 3181911
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