Bioengineering of bacteria to assemble custom-made polyester affinity resins
Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on...
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Veröffentlicht in: | Applied and Environmental Microbiology 2015-01, Vol.81 (1), p.282-291 |
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description | Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. |
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This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>EISSN: 1098-6596</identifier><identifier>DOI: 10.1128/AEM.02595-14</identifier><identifier>PMID: 25344238</identifier><identifier>CODEN: AEMIDF</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Bacteria ; Bacteria - genetics ; Bacteria - growth & development ; Bacteria - metabolism ; Biotechnology ; Chromatography ; Chromatography, Affinity ; Fermentation ; Metabolic Engineering - methods ; Polyesters - metabolism ; Protein Binding ; Proteins ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Resins</subject><ispartof>Applied and Environmental Microbiology, 2015-01, Vol.81 (1), p.282-291</ispartof><rights>Copyright © 2015, American Society for Microbiology. All Rights Reserved.</rights><rights>Copyright American Society for Microbiology Jan 2015</rights><rights>Copyright © 2015, American Society for Microbiology. All Rights Reserved. 2015 American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c445t-6aa575befb7190552ae3f9c3d2b5e0d84d7ae7e61f23978cf76b1b563f90b19d3</citedby><cites>FETCH-LOGICAL-c445t-6aa575befb7190552ae3f9c3d2b5e0d84d7ae7e61f23978cf76b1b563f90b19d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272739/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272739/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,3175,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25344238$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Nojiri, H.</contributor><creatorcontrib>Hay, Iain D</creatorcontrib><creatorcontrib>Du, Jinping</creatorcontrib><creatorcontrib>Burr, Natalie</creatorcontrib><creatorcontrib>Rehm, Bernd H A</creatorcontrib><title>Bioengineering of bacteria to assemble custom-made polyester affinity resins</title><title>Applied and Environmental Microbiology</title><addtitle>Appl Environ Microbiol</addtitle><description>Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." 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subjects | Bacteria Bacteria - genetics Bacteria - growth & development Bacteria - metabolism Biotechnology Chromatography Chromatography, Affinity Fermentation Metabolic Engineering - methods Polyesters - metabolism Protein Binding Proteins Recombinant Proteins - genetics Recombinant Proteins - metabolism Resins |
title | Bioengineering of bacteria to assemble custom-made polyester affinity resins |
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