Conditionally active biological proteins
A conditional activity and biological protein technology, applied in the field of protein evolution and activity, can solve problems such as protein instability
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[0358] Monoclonal antibodies can be produced using any technique that provides antibodies produced by continuous cell line culture. Examples include hybridoma technology, trisomy hybridoma technology, human B-cell hybridoma technology and Epstein-Barr virus hybridoma technology (see for example, Cole (1985), Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy), Alan R . Liss, Inc., pp. 77-96).
[0359] Techniques described for the production of single chain antibodies (see eg, US Pat. No. 4,946,778) can be adapted to produce single chain antibodies directed against polypeptides of the invention. Alternatively, transgenic mice expressing humanized antibodies against these polypeptides or fragments thereof can be used. Antibodies raised against polypeptides of the invention can be used to screen for similar polypeptides (eg, enzymes) from other organisms and samples. In these techniques, polypeptides from an organism are contacted with antibodies...
Embodiment 1
[0434] Example 1: Overview of Multiwall Assays (eg, 96-well Assays) for Temperature Abruptions
[0435] Fluorescent substrates are added to each well of the multi-wall plate for an appropriate time at either wild-type or a new lower reaction temperature (eg, 37°C or 25°C as described above). Fluorescence is detected by measuring fluorescence in a fluorescent microplate reader under appropriate excitation and emission spectra (eg, 320 nm excitation / 405 nm emission). Measure relative fluorescence units (RFU). Supernatants obtained from wild-type molecule and plasmid / vector transformed cells served as positive and negative controls. Duplicate reactions were performed for each sample, each reaction temperature, and positive and negative controls.
[0436] are active at lower temperatures (e.g., mutants are active at 25°C), and are less active at wild-type temperatures (e.g., 10%, 20%, 30%, 40% or more active at 37°C) more), and thus a mutant with an activity ratio greater than ...
Embodiment 2
[0437] Example 2: Overview of Different Assays (eg, 14-mL Assay) for Determining Activity of Temperature Mutants
[0438] Mutants of identified major temperature-sensitive shoots were expressed in 14 ml culture tubes and their enzymatic activities were assayed at wild-type (eg, 37°C) and lower temperatures (eg, 25°C). Proteins were expressed and purified as described above for the multiwall method, except expression was done in a different format (14 ml tubes) rather than the multiwall (96-well plate) format.
[0439] The supernatant of each mutant is transferred to a multi-walled plate, such as a 96-well plate. Fluorescent substrate was added to each tube at the indicated reaction temperature (wild type, lower temperature) for the desired period of time. Wild-type molecules were used as positive controls and supernatants from cells transformed with vector only were used as negative controls. Fluorescence is detected by measuring fluorescence in a fluorescent microplate read...
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