Document Detail

Optimized expression of the dirigent protein AtDIR6 in Pichia pastoris and impact of glycosylation on protein structure and function.
MedLine Citation:
PMID:  23188459     Owner:  NLM     Status:  Publisher    
Phenoxy radical coupling reactions are involved in the biosynthesis of lignans in planta. Interestingly, the reaction can be guided by dirigent proteins, which mediate the stereoselective formation of either (+) or (-)-pinoresinol from coniferyl alcohol. So far, the mechanism is poorly understood, and for detailed mechanistic studies, a heterologous expression platform which allows the cost-effective, fast, and robust expression in high yields is needed. We established a reliable, high-yield fed-batch fermentation process with Pichia pastoris resulting in 47 mg L(-1) of the dirigent protein AtDIR6, which represents a more than 250-fold increase compared to previous studies. Biochemical characterization of AtDIR6 produced with P. pastoris showed an overall agreement in protein structure, N-glycosylation sites, and dirigent activity compared to AtDIR6 produced by plant cell cultures of Solanum peruvianum. CD spectroscopy verified the β-barrel structure proposed by earlier studies and bioconversion experiments revealed similar activities to plant-derived protein, validating P. pastoris as a suitable expression system for dirigent proteins. Compared to the complex glycan structures of most plant cells, proteins produced with P. pastoris have the advantage that they can be enzymatically deglycosylated under non-denaturating conditions. With this study, we demonstrate that the glycan structures of AtDIR6 are essential for structure, solubility, and function of the protein as deglycosylation induced conformational changes leading to the complete loss in dirigent activity and subsequent protein aggregation.
Christian Kazenwadel; Janosch Klebensberger; Sven Richter; Jens Pfannstiel; Uwe Gerken; Benjamin Pickel; Andreas Schaller; Bernhard Hauer
Related Documents :
24872809 - Mass spectrometry-based n-glycoproteomics for cancer biomarker discovery.
23551859 - Distinct functions of nuclear distribution proteins lis1, ndel1 and nudcl in regulating...
16029889 - Parathion effects on protein synthesis in the seminiferous tubules of mice.
24858899 - Photosynthetic and molecular responses of the marine diatom thalassiosira pseudonana to...
3917999 - Restoration of vegetative penicillin-binding proteins during germination and outgrowth ...
24752279 - Levels of germination proteins in bacillus subtilis dormant, superdormant, and germinat...
18429089 - Strategies for protein purification.
21737499 - Aeromonas hydrophila moty is essential for polar flagellum function, requires coordinat...
7651829 - A variety of dna-binding and multimeric proteins contain the histone fold motif.
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-11-28
Journal Detail:
Title:  Applied microbiology and biotechnology     Volume:  -     ISSN:  1432-0614     ISO Abbreviation:  Appl. Microbiol. Biotechnol.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8406612     Medline TA:  Appl Microbiol Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Institute of Technical Biochemistry, University of Stuttgart, D-70569, Stuttgart, Germany.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Previous Document:  Adaptation of the Spodoptera exigua Se301 insect cell line to grow in serum-free suspended culture. ...
Next Document:  Response of activated sludge to the treatment of oxytetracycline production waste stream.