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Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
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2010 (Engelska)Ingår i: PLANT BIOTECHNOLOGY, ISSN 1342-4580, Vol. 27, nr 3, s. 251-258Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as N-glycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119-138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.

Ort, förlag, år, upplaga, sidor
2010. Vol. 27, nr 3, s. 251-258
Nyckelord [en]
Pichia pastoris, plant protein expression, xyloglucan endo-transglycosylase/hydrolase (XTH) genes, XET, XEH
Nationell ämneskategori
Växtbioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-28349DOI: 10.5511/plantbiotechnology.27.251ISI: 000280085800006Scopus ID: 2-s2.0-77953215212OAI: oai:DiVA.org:kth-28349DiVA, id: diva2:387077
Konferens
Colloquium on Green Chemistry in Plants and Microorganisms Japanese Soc Promot Sci, Stockholm, SWEDEN, MAY 25, 2009
Anmärkning
QC 20110113Tillgänglig från: 2011-01-13 Skapad: 2011-01-13 Senast uppdaterad: 2020-03-05Bibliografiskt granskad
Ingår i avhandling
1. In vitro and in vivo approaches in the characterization of XTH gene products
Öppna denna publikation i ny flik eller fönster >>In vitro and in vivo approaches in the characterization of XTH gene products
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

ABSTRACT

The xyloglucan endo-transglycosylase/hydrolase (XTH) genes are found in all vascular and some nonvascular plants. The XTH genes encode proteins which comprise a subfamily of glycoside hydrolase (GH) family 16 in the Carbohydrate-Active enZYmes (CAZY) classification. The XTH gene products are believed to play intrinsic role in cell wall modification during growth and development throughout the lifetime of the plant. In the present investigation, biochemical and reverse genetic approaches were used to better understand the functions of individual members of the XTH gene family of two important plants: the model organism Arabidopsis thaliana and the grain crop barley (Hordeum vulgare). A phylogenetic tree of the xyloglucan-active enzymes of GH16 has previously been constructed, where enzymes with similar activities have been shown to cluster together. Several members of phylogenetic Group I/II and III-B, predicted to exhibit xyloglucan endo-transglycosylase activity (EC 2.4.1.207) and members of Group III-A, predicted to exhibit xyloglucan endo-hydrolase activity (EC 3.2.1.151), were included to analyze the functional diversity of XTH gene products. A heterologous expression system using the yeast Pichia pastoris was found to be effective for recombinant protein production with a success rate of ca. 50%. XTH gene products were obtained in soluble and active forms for subsequent biochemical characterization.

In order to be able to screen larger numbers of protein producing clones, a fast and easy method is required to identify clones expressing active protein in high enough amounts. Thus, a miniaturized XET/XEH assay for high-throughput analysis was developed, which was able to identify activities with good precision and with a reduced time and materials consumption and a reduced work load.

Enzyme kinetic analysis indicated that the XET or XEH activity of all XTH gene products characterized in the present study corresponded to predictions based on the previously revised phylogenetic clustering. To gain insight into the biological function of the predominant XEHs AtXTH31 and AtXTH32, which are highly expressed in rapidly developing tissues, a reverse genetic approach was employed using T-DNA insertion lines of the A. thaliana Columbia ecotype. Genotypic and phenotypic characterization, together with in situ assays of XET and XEH activities, in single- and double-knock-out mutants indicated that these Group III-A enzymes are active in expanding tissues of the A. thaliana roots and hypocotyl.  Although suppression of in muro XEH activity was clearly observed in the double-knock-out, no significant growth phenotype was observed, with the exception that radicle emergence appeared to be faster than in the wild type plants.

Keywords: Arabidopis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-transglycosylase, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-throughput screening

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2011. s. x, 48
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011: 1
Nyckelord
Arabiodopsis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-througput screening
Nationell ämneskategori
Botanik Biokemi och molekylärbiologi Biokemi och molekylärbiologi
Forskningsämne
SRA - Molekylär biovetenskap
Identifikatorer
urn:nbn:se:kth:diva-28222 (URN)978-91-7415-848-9 (ISBN)
Disputation
2011-02-02, FB54, Roslagstullsbacken 21, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20110114Tillgänglig från: 2011-01-14 Skapad: 2011-01-12 Senast uppdaterad: 2011-01-14Bibliografiskt granskad

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