Arabidopsis thaliana Recombinants
The production and isolation of recombinant proteins are key steps in modern molecular biology. Expression vectors and platforms have been used for a variety of hosts, including both prokaryotic and eukaryotic systems. In basic plant research, Arabidopsis thaliana recombinant is the central model for which a large number of genetic and genomic resources are available, and enormous knowledge has been accumulated in recent years, especially since the elucidation of its genome in 2000.
However, until recently an Arabidopsis platform was lacking for preparative-scale production of homologous recombinant proteins. We recently established an Arabidopsis-based superexpression system and used it for a pilot structural study of an integral multi-subunit membrane protein complex. This review summarizes the benefits and added potential of the model plant system for protein production.
Materials And Methods
- Growing conditions and plants
Both wild-type and ptd mutant plants carrying a transferred DNA (T-DNA) insert, SALK_127477 (ptd-1) and SAIL_567_D09 (ptd-2), were of the Columbia ecotype unless otherwise noted. All plants were grown under long-day conditions (16 h day and 8 h night) in a culture room at a mean temperature of 18–22 °C or in a greenhouse.
- Phenotypic analysis
Plants were photographed with a Sony DSC-F828 digital camera (Sony, Tokyo, Japan). Dissected tetrads were stained with 0.01% toluidine blue. Pollen grains were stained with Alexander’s solution (Alexander, 1969) to test pollen viability. Images were obtained using a Nikon dissecting microscope (Nikon, Tokyo, Japan) with an Optronics digital camera (Optronics, Goleta, CA). Wild-type and mutant inflorescences were harvested, and chromosome spreads were prepared as described by Ross et al. (1996) and stained with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI).
Images of chromosome spreads were obtained using a Nikon Eclipse E800 microscope (Nikon) and a Hamamatsu digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Images were edited using Photoshop 7.0 (Adobe Systems, Mountain View, CA). The number of chiasmata was counted from diakinesis images in a similar way as described previously (Sanchez Moran et al., 2001). Transmission electron microscopy was carried out as previously described (Li et al., 2004). All statistical analyzes were carried out as previously described (Zar, 1974).
- PCR and sequencing of T-DNA insertion sites
Genomic DNAs were extracted from rosette leaves using 2x CTAB buffer [2% (wt/vol) cetyltrimethylammonium bromide, 1.4 M NaCl, 100 Tris-HCl mM, pH 8.0 and 20 mM EDTA].
PCR was used to identify plants that were homozygous and heterozygous for one of two T-DNA insertions, SALK_127477 (ptd-1) and SAIL_567_D09 (ptd-2). For the SALK_127447 line, the wild-type allele was amplified with primers oMC1607 and oMC1608, and the mutant allele was amplified with oMC1607 and oMC1863, a primer specific for the left border of the T-DNA. For the SAIL_567_D09 line, the wild-type allele was amplified using oMC1911 and oMC1912, and the mutant allele was amplified using a gene-specific primer oMC1911 and the T-DNA left border primer oMC2009.
The oMC1607/oMC1863 or oMC1911/oMC2009 products were purified and sequenced to confirm their identity.
Nb, Nicotiana benthamiana; OT, oligosaccharyltransferase
Arabidopsis, recombinant proteins, protein N-glycosylation, complex-type N-glycans, endoplasmic reticulum, Golgi apparatus, plasma membrane, trans-Golgi network