High-throughput screening of expressed DNA libraries in filamentous fungi
First Claim
1. A method of producing a plurality of proteins having an activity or property of interest encoded by a library of DNA vectors, wherein the library of vectors comprises a plurality of different vectors, each different vector comprising a different protein-encoding nucleic acid sequence, said nucleic acid sequence being operably linked to an expression-regulating region and optionally a secretion signal encoding sequence, the method comprising the steps of:
- (a) stably transforming a plurality of individual filamnentous fungi, wherein the fungi are selected from the group consisting of Aspergillus, Fusarium, Chrysosporium and Trichoderma, said fungi having a phenotype characterized by growth in suspension and by the production of transferable reproductive elements which are monoclonal and readily dispersed in suspension, with said library of DNA vectors so as to introduce into each of the plurality of the individual fungi at least one protein-encoding nucleic acid sequence;
(b) culturing the transformed mutant filamentous fungi under conditions conducive to formation of transferable reproductive elements which are monoclonal and readily dispersed in suspension;
(c) separating from one another a plurality of transferable reproductive elements in suspension;
(d) transferring the separated transferable reproductive elements to secondary cultures; and
(e) culturing into monoclonal cultures or monoclonal colonies the individual transferable reproductive elements in said secondary cultures, under conditions condacive to expression of the proteins encoded by the protein-encoding nucleic acid sequences.
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Abstract
The invention provides a method for the expression of exogenous DNA libraries in filamentous fungi. The fungi are capable of processing intron-containing eukaryotic genes, and also can carry out post-translational processing steps such as glyclosylation and protein folding. The invention provides for the use of fungi with altered morphology, which permits high-throughput screening and directed molecular evolution of expressed proteins. The same transformed fungi may be used to produce larger quantities of protein for isolation, characterization, and application testing, and may be suitable for commercial production of the protein as well.
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Citations
16 Claims
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1. A method of producing a plurality of proteins having an activity or property of interest encoded by a library of DNA vectors, wherein the library of vectors comprises a plurality of different vectors, each different vector comprising a different protein-encoding nucleic acid sequence, said nucleic acid sequence being operably linked to an expression-regulating region and optionally a secretion signal encoding sequence, the method comprising the steps of:
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(a) stably transforming a plurality of individual filamnentous fungi, wherein the fungi are selected from the group consisting of Aspergillus, Fusarium, Chrysosporium and Trichoderma, said fungi having a phenotype characterized by growth in suspension and by the production of transferable reproductive elements which are monoclonal and readily dispersed in suspension, with said library of DNA vectors so as to introduce into each of the plurality of the individual fungi at least one protein-encoding nucleic acid sequence; (b) culturing the transformed mutant filamentous fungi under conditions conducive to formation of transferable reproductive elements which are monoclonal and readily dispersed in suspension; (c) separating from one another a plurality of transferable reproductive elements in suspension; (d) transferring the separated transferable reproductive elements to secondary cultures; and (e) culturing into monoclonal cultures or monoclonal colonies the individual transferable reproductive elements in said secondary cultures, under conditions condacive to expression of the proteins encoded by the protein-encoding nucleic acid sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification