Improved Assembly of Multimeric Genes for the Biosynthetic Production of Protein Polymers
We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach...
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| Veröffentlicht in: | Biomacromolecules 2002-07, Vol.3 (4), p.874-879 |
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| container_title | Biomacromolecules |
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| creator | Goeden-Wood, Nichole L Conticello, Vincent P Muller, Susan J Keasling, Jay D |
| description | We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the β-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI−TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a β-sheet structure in aqueous solution. |
| doi_str_mv | 10.1021/bm0255342 |
| format | Article |
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Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the β-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI−TOF mass spectrometry. 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Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the β-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI−TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a β-sheet structure in aqueous solution.</description><subject>Amino Acid Sequence</subject><subject>Biopolymers - biosynthesis</subject><subject>Biopolymers - genetics</subject><subject>Cloning, Molecular</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Genes, Synthetic - genetics</subject><subject>Protein Biosynthesis</subject><subject>Protein Engineering - methods</subject><subject>Protein Structure, Secondary</subject><subject>Proteins - genetics</subject><subject>Tandem Repeat Sequences</subject><issn>1525-7797</issn><issn>1526-4602</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kDFPwzAQhS0EolAY-AMoCyCGwNmx43gsFZRKRXSAgSlKnItIlcTFTpDy73FpBQtiune67z3pHiFnFG4oMHqbN8CEiDjbI0dUsDjkMbD9by1CKZUckWPnVgCgIi4OyYgyUCqJ5BF5mzdraz6xCCbOYZPXQ2DK4Kmvu6pBW-lghi26oDQ26N4xuKuMG1qvOn9aWlP0uqtMu_H4rcOqDZamHrzVnZCDMqsdnu7mmLw-3L9MH8PF82w-nSzCjIPqwpyVAgoUApSmMkaMOY0AE5opphF1xkpOc1nwTComIC9BcIx1gRFwEYkyGpOrba7_46NH16VN5TTWddai6V0qBRc8kQl48vJ_kiZSUhV78HoLamucs1ima1s1mR1SCumm8fSncc-e70L7vMHil9xV7IGLLZBpl65Mb1vfxh9BX3WZhyY</recordid><startdate>20020701</startdate><enddate>20020701</enddate><creator>Goeden-Wood, Nichole L</creator><creator>Conticello, Vincent P</creator><creator>Muller, Susan J</creator><creator>Keasling, Jay D</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20020701</creationdate><title>Improved Assembly of Multimeric Genes for the Biosynthetic Production of Protein Polymers</title><author>Goeden-Wood, Nichole L ; Conticello, Vincent P ; Muller, Susan J ; Keasling, Jay D</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a409t-b2f50de5509c176ee64130e81a92ceeca2f41b7d4a79250bf054e6cde304535f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Amino Acid Sequence</topic><topic>Biopolymers - biosynthesis</topic><topic>Biopolymers - genetics</topic><topic>Cloning, Molecular</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Genes, Synthetic - genetics</topic><topic>Protein Biosynthesis</topic><topic>Protein Engineering - methods</topic><topic>Protein Structure, Secondary</topic><topic>Proteins - genetics</topic><topic>Tandem Repeat Sequences</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Goeden-Wood, Nichole L</creatorcontrib><creatorcontrib>Conticello, Vincent P</creatorcontrib><creatorcontrib>Muller, Susan J</creatorcontrib><creatorcontrib>Keasling, Jay D</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Biomacromolecules</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Goeden-Wood, Nichole L</au><au>Conticello, Vincent P</au><au>Muller, Susan J</au><au>Keasling, Jay D</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Improved Assembly of Multimeric Genes for the Biosynthetic Production of Protein Polymers</atitle><jtitle>Biomacromolecules</jtitle><addtitle>Biomacromolecules</addtitle><date>2002-07-01</date><risdate>2002</risdate><volume>3</volume><issue>4</issue><spage>874</spage><epage>879</epage><pages>874-879</pages><issn>1525-7797</issn><eissn>1526-4602</eissn><abstract>We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the β-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI−TOF mass spectrometry. 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| language | eng |
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| source | MEDLINE; American Chemical Society Journals |
| subjects | Amino Acid Sequence Biopolymers - biosynthesis Biopolymers - genetics Cloning, Molecular Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Genes, Synthetic - genetics Protein Biosynthesis Protein Engineering - methods Protein Structure, Secondary Proteins - genetics Tandem Repeat Sequences |
| title | Improved Assembly of Multimeric Genes for the Biosynthetic Production of Protein Polymers |
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