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 (ABPI), 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. Conclusion: 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.

Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells coexpressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the reinfection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.

Derivation of an urban materials spectral library through emittance and reflectance spectroscopy

Recent advances in thermal infrared remote sensing include the increased availability of airborne hyperspectral imagers (such as the Hyperspectral Thermal Emission Spectrometer, HyTES, or the Telops HyperCam and the Specim aisaOWL), and it is planned that an increased number spectral bands in the long-wave infrared (LWIR) region will soon be measured from space at reasonably high spatial resolution (by imagers such as HyspIRI). Detailed LWIR emissivity spectra are required to best interpret the observations from such systems. This includes the highly heterogeneous urban environment, whose construction materials are not yet particularly well represented in spectral libraries. Here, we present a new online spectral library of urban construction materials including LWIR emissivity spectra of 74 samples of impervious surfaces derived using measurements made by a portable Fourier Transform InfraRed (FTIR) spectrometer. FTIR emissivity measurements need to be carefully made, else they are prone to a series of errors relating to instrumental setup and radiometric calibration, which here relies on external blackbody sources. The performance of the laboratory-based emissivity measurement approach applied here, that in future can also be deployed in the field (e.g. to examine urban materials in situ), is evaluated herein. Our spectral library also contains matching short-wave (VIS–SWIR) reflectance spectra observed for each urban sample. This allows us to examine which characteristic (LWIR and) spectral signatures may in future best allow for the identification and discrimination of the various urban construction materials, that often overlap with respect to their chemical/mineralogical constituents. Hyperspectral or even strongly multi-spectral LWIR information appears especially useful, given that many urban materials are composed of minerals exhibiting notable reststrahlen/absorption effects in this spectral region. The final spectra and interpretations are included in the London Urban Micromet data Archive (LUMA; http://LondonClimate.info/LUMA/SLUM.html).