Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries
| Autor: | Paul W. King, Scott Plummer, Michael Seibert, Dianne Ahmann, Jonathan E. Meuser, Maria L. Ghirardi, Lauren E. Nagy, Matthew C. Posewitz |
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| Rok vydání: | 2006 |
| Předmět: |
Iron-Sulfur Proteins
Hydrogenase Bioengineering medicine.disease_cause Protein Engineering Applied Microbiology and Biotechnology law.invention chemistry.chemical_compound Hyda law Peptide Library medicine Escherichia coli Gene Clostridium biology Active site DNA Shuffling General Medicine biology.organism_classification Recombinant Proteins chemistry Biochemistry biology.protein Recombinant DNA Heterologous expression DNA Biotechnology |
| Zdroj: | Biotechnology letters. 29(3) |
| ISSN: | 1573-6776 |
| Popis: | A gene-shuffling technique was identified, optimized and used to generate diverse libraries of recombinant [FeFe]-hydrogenases. Six native [FeFe]-hydrogenase genes from species of Clostridia were first cloned and separately expressed in Escherichia coli concomitantly with the assembly proteins required for [FeFe]-hydrogenase maturation. All enzymes, with the exception of C. thermocellum HydA, exhibited significant activity when expressed. Single-stranded DNA fragments from genes encoding the two most active [FeFe]-hydrogenases were used to optimize a gene-shuffling protocol and generate recombinant enzyme libraries. Random sampling demonstrates that several shuffled products are active. This represents the first successful application of gene-shuffling using hydrogenases. Moreover, we demonstrate that a single set of [FeFe]-hydrogenase maturation proteins is sufficient for the heterologous assembly of the bioinorganic active site of several native and shuffled [FeFe]-hydrogenases. |
| Databáze: | OpenAIRE |
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