Display of a maize cDNA library on baculovirus infected insect cells.
BACKGROUND
Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings.


RESULTS
We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins.


CONCLUSIONS
The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.
Meller Harel
Helene Y
HY
Faculty of biological sciences, University of Leeds, Leeds, LS2 9JT, UK. bmbhymh@leeds.ac.uk
Fontaine
Veronique
V
Chen
Hongying
H
Jones
Ian M
IM
Millner
Paul A
PA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D037282:Calreticulin / Q000096:biosynthesis; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D008280:Magnetics; D010940:Plant Proteins / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D018411:Spodoptera / Q000378:metabolism / Q000821:virology; D014162:Transfection; D003313:Zea mays / Q000235:genetics*
D037282:Calreticulin; D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C083846:auxin-binding protein 1