Multiplexed expression and screening for recombinant protein production in mammalian cells

Abstract Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.</p

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

High throughput generation and characterization of recombinant antibodies

<p><b>Copyright information:</b></p><p>Taken from "Application of phage display to high throughput antibody generation and characterization"</p><p>http://genomebiology.com/2007/8/11/R254</p><p>Genome Biology 2007;8(11):R254-R254.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2258204.</p><p></p> For each target antigen, 94 clones were screened in ELISA and sequenced to identify unique clones. The plot shows the number of antigens selected (x-axis) versus the number of unique positive antibodies generated (y-axis) for each antigen. Separate plots are presented for antigens produced in either bacterial or mammalian expression systems, illustrating the improved success rate for mammalian antigens. Example specificity data for antibodies selected against Slam f9 (produced in the HEK293 mammalian expression system). All antibodies are screened against target antigen, the relevant fusion partner that was used in selection, keyhole limpet hemocyanin (KLH), thyroglobulin, myoglobin, cytochrome c, human IgG, laminin, fibronectin, α-glycerol phosphate dehydrogenase, and total protein lysates from zebra fish () and yeast (). Results are shown for 22 different antibodies as well as our routine anti-desmin control (des-D7) and a no antibody control. Detection was via time resolved fluorescence and values are shown on a logarithmic scale. Global summary of secondary ELISA data for all antibodies in secondary screening. Signal generated on specific antigen is shown for all 4,437 samples (solid block). Signal achieved on one of the ten irrelevant antigens (cytochrome c) is also shown

Frequency of VH and V kappa/V lambda germline gene combinations selected from the library

<p><b>Copyright information:</b></p><p>Taken from "Application of phage display to high throughput antibody generation and characterization"</p><p>http://genomebiology.com/2007/8/11/R254</p><p>Genome Biology 2007;8(11):R254-R254.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2258204.</p><p></p> High quality sequence of all 4,437 clones undergoing secondary screening was generated by sequencing with six primers covering the VH and VL gene segments in both forward and reverse orientations. A consensus sequence was generated and the most closely related antibody germ line genes were identified. Frequency of different combinations of VH and V kappa/V lambda germ-line genes occurring in the selected antibodies, represented both numerically and by color coding. Frequency of different combinations of VH and V kappa/V lambda germline genes among the selected antibodies

Detection sensitivity in a bead-based performance assay for a panel of antigens

<p><b>Copyright information:</b></p><p>Taken from "Application of phage display to high throughput antibody generation and characterization"</p><p>http://genomebiology.com/2007/8/11/R254</p><p>Genome Biology 2007;8(11):R254-R254.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2258204.</p><p></p> The sensitivity limits of 90 antibodies to 9 different antigens were tested using a mix of antigen coated beads of different antigen densities. To summarize these data, the relative median fluorescent intensity on the bead comprising 459,000 antigen copies per bead was calculated (actual values and clone IDs given in Additional data file 2) and the ratio relative to uncoated beads plotted. To further illustrate sensitivity achieved, the identity of the lowest density bead that could be detected is indicated by the data label according to the following guide: 459,000 antigen copies per bead only (diamonds), down to 57,000 antigen copies per bead (square), and down to 18,000 antigen copies per bead (triangles). The antigens were: 1, Efna2; 2, Efna4; 3, Plaur; 4, Alcam; 5, Il13ra1; 6, Sigrr; 7, Ngfr; 8, Cd22; and 9, Vcam1