Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis
If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavag...
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| Veröffentlicht in: | The Journal of biological chemistry 1991-11, Vol.266 (32), p.21355-21357 |
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| container_issue | 32 |
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| container_title | The Journal of biological chemistry |
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| creator | WALLACE, C. J. A GUILLEMETTE, J. G HIBIYA, Y SMITH, M |
| description | If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be
achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome
c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively,
bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified
fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation,
for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic
ones, that are appropriate to the semisynthetic goals. |
| doi_str_mv | 10.1016/S0021-9258(18)54643-3 |
| format | Article |
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achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome
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bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified
fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation,
for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic
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achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome
c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively,
bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified
fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation,
for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic
ones, that are appropriate to the semisynthetic goals.</description><subject>Amino Acid Sequence</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Cyanogen Bromide</subject><subject>Cytochrome c Group - chemical synthesis</subject><subject>Cytochrome c Group - genetics</subject><subject>Cytochrome c Group - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>General aspects, investigation methods</subject><subject>Horses</subject><subject>improvements</subject><subject>Methionine</subject><subject>methodology</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Oligodeoxyribonucleotides</subject><subject>Oxidoreductases - metabolism</subject><subject>Peptide Fragments - isolation & purification</subject><subject>Protein Conformation</subject><subject>Protein Engineering - methods</subject><subject>Proteins</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae - metabolism</subject><subject>Serine</subject><subject>site-directed mutagenesis</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkMtKxDAUhoMo43h5BKELEV1Uc2-7FPEGggsdcBdOk9NppE3HpoPM29s6g2aTcP7vJCcfIWeMXjPK9M0bpZylBVf5JcuvlNRSpGKPzBnNx4NiH_tk_occkqMYP-m4ZMFmZMa0ygpN52RxH2oI1odlsuq7AX1IMCx9QOynmoUVlL7xg8eYlJvEdm3pw5S06wGWGDD6mEBwScTWx00Y6qlyQg4qaCKe7vZjsni4f797Sl9eH5_vbl9SKyUdUg2gc-5yW6kqczxzUObWaVehUkJrDii14Aqkk1wUQF3BMiokK7MCFAKIY3KxvXec_WuNcTDjEBabBgJ262iYFlrmVI2g2oK272LssTKr3rfQbwyjZtJpfnWayZVhufnVacTYd7Z7YF226P67tv7G_HyXQ7TQVP3kMv5himqpMvGP1X5Zf_seTek7W2NruNZGcMOZGL_8AzmEipc</recordid><startdate>19911115</startdate><enddate>19911115</enddate><creator>WALLACE, C. 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Psychology</topic><topic>General aspects, investigation methods</topic><topic>Horses</topic><topic>improvements</topic><topic>Methionine</topic><topic>methodology</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Oligodeoxyribonucleotides</topic><topic>Oxidoreductases - metabolism</topic><topic>Peptide Fragments - isolation & purification</topic><topic>Protein Conformation</topic><topic>Protein Engineering - methods</topic><topic>Proteins</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae - metabolism</topic><topic>Serine</topic><topic>site-directed mutagenesis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>WALLACE, C. J. A</creatorcontrib><creatorcontrib>GUILLEMETTE, J. 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G</au><au>HIBIYA, Y</au><au>SMITH, M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1991-11-15</date><risdate>1991</risdate><volume>266</volume><issue>32</issue><spage>21355</spage><epage>21357</epage><pages>21355-21357</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be
achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome
c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively,
bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified
fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation,
for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic
ones, that are appropriate to the semisynthetic goals.</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>1657960</pmid><doi>10.1016/S0021-9258(18)54643-3</doi><tpages>3</tpages><oa>free_for_read</oa></addata></record> |
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| language | eng |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Analytical, structural and metabolic biochemistry Animals Base Sequence Biological and medical sciences Cyanogen Bromide Cytochrome c Group - chemical synthesis Cytochrome c Group - genetics Cytochrome c Group - metabolism Fundamental and applied biological sciences. Psychology General aspects, investigation methods Horses improvements Methionine methodology Molecular Sequence Data Mutagenesis, Site-Directed Oligodeoxyribonucleotides Oxidoreductases - metabolism Peptide Fragments - isolation & purification Protein Conformation Protein Engineering - methods Proteins Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - metabolism Serine site-directed mutagenesis |
| title | Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis |
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