Document Detail


Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris.
MedLine Citation:
PMID:  17317217     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The Nicotiana tabacum lectin, also called Nictaba, is a nucleocytoplasmic plant lectin expressed in tobacco leaves after exposure to jasmonates. Purification of the lectin from raw material is a time-consuming process, demanding large amounts of induced plant material. In addition, the lectin yield is low and purified lectin fractions are always contaminated with low molecular weight compounds such as phenols. In a way to improve and facilitate the purification of the tobacco lectin, we cloned the Nictaba gene in a vector optimized for protein expression in the methylotrophic yeast Pichia pastoris. In this report, we present data of the expression profile of recombinant Nictaba in the P. pastoris culture medium and in P. pastoris cells together with the purification strategy using ion exchange chromatography and affinity chromatography on a column with immobilized ovomucoid. Pichia transformants were estimated to express approximately 6mg of recombinant lectin per liter medium after a 72h culture. SDS-PAGE and Western blot analysis revealed that the recombinant lectin expressed in Pichia exists in two molecular forms. Edman degradation and mass spectrometry analysis confirmed the presence of at least two forms of recombinant lectin with molecular weights of 19,060 and 20,100Da, corresponding to lectin polypeptides similar to the fully processed Nictaba which is N-terminally blocked, and Nictaba extended at the N-terminus with the amino acids residues EAEAYVEFT due to incomplete processing of the alpha-factor mating sequence. Further characterisation of the recombinant lectin revealed agglutination and carbohydrate-binding properties similar to the native tobacco lectin.
Authors:
Nausicaä Lannoo; Wouter Vervecken; Paul Proost; Pierre Rougé; Els J M Van Damme
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-01-20
Journal Detail:
Title:  Protein expression and purification     Volume:  53     ISSN:  1046-5928     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2007 Jun 
Date Detail:
Created Date:  2007-03-27     Completed Date:  2007-07-19     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  275-82     Citation Subset:  IM    
Affiliation:
Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Base Sequence
Chromatography, Affinity
Chromatography, Ion Exchange
Cloning, Molecular
DNA, Plant / genetics
Gene Expression Profiling
Genes, Plant
Genetic Vectors
Molecular Sequence Data
Pichia / genetics*
Plant Lectins / genetics*,  isolation & purification*
Plasmids / genetics
Recombinant Proteins / genetics,  isolation & purification
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tobacco / genetics*
Transformation, Genetic
Chemical
Reg. No./Substance:
0/DNA, Plant; 0/Plant Lectins; 0/Recombinant Proteins

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