A robust and reliable method for high yield deuterated recombinant protein production using Escherichia coli BL21.

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dc.contributor.authorDuff, APen_AU
dc.contributor.authorWilde, KLen_AU
dc.contributor.authorRekas, Aen_AU
dc.contributor.authorLake, Ven_AU
dc.contributor.authorHolden, PJen_AU
dc.date.accessioned2021-08-17T02:37:25Zen_AU
dc.date.available2021-08-17T02:37:25Zen_AU
dc.date.issued2017-07-12en_AU
dc.date.statistics2021-08-10en_AU
dc.description.abstractWe have developed a method that has proven highly reliable for the deuteration of a broad range of proteins by recombinant expression in Escherichia coli BL21. Typical biomass yields are 40-80 g/L wet weight, yielding 50-400mg/L purified protein. This method uses a simple, relatively inexpensive defined medium, and routinely results in a high yield expression without need for optimisation. The key elements are: very tight control of expression, careful starter culture adaption steps, and strict maintenance of aerobic conditions ensuring exponential growth. Temperature is reduced as required to prevent biological oxygen demand exceeding maximum aeration capacity. Glycerol is the sole carbon source. We have not encountered an upper limit for the size of proteins that can be expressed, achieving excellent expression for proteins from 7-112 kDa and the quantity produced at 1L scale ensures that no SANS, NMR or neutron crystallography experiment is limited by the amount of deuterated material. Where difficulties remain, these tend to be cases of protein solubility exacerbated by high protein concentration and slightly increased stickiness of proteins in D O. There are some very few cases in which we have been unable to express a protein by our method despite unlabelled expression being reliable in rich media using induction at low OD. Few proteins tested have not expressed in deuterated medium despite unlabelled expression being reliable.en_AU
dc.identifier.citationDuff, A., Wilde, K., Rekas, A., Lake, V., & Holden, P. (2017). A robust and reliable method for high yield deuterated recombinant protein production using Escherichia coli BL21. Paper presented at ICNS 2017 (International Conference on Neutron Scattering), Daejeon, South Korea, 9 to 13 July 2017. Retrieved from: http://www.icns2017.org/program.phpen_AU
dc.identifier.conferenceenddate13 July 2017en_AU
dc.identifier.conferencenameICNS 2017 (International Conference on Neutron Scattering)en_AU
dc.identifier.conferenceplaceDaejeon, South Koreaen_AU
dc.identifier.conferencestartdate9 July 2017en_AU
dc.identifier.urihttp://www.icns2017.org/program.phpen_AU
dc.identifier.urihttps://apo.ansto.gov.au/dspace/handle/10238/11376en_AU
dc.language.isoenen_AU
dc.publisherInternational Conference on Neutron Scatteringen_AU
dc.subjectDeuterationen_AU
dc.subjectProteinsen_AU
dc.subjectEscherichia colien_AU
dc.subjectGlycerolen_AU
dc.subjectSolubilityen_AU
dc.subjectCrystallographyen_AU
dc.titleA robust and reliable method for high yield deuterated recombinant protein production using Escherichia coli BL21.en_AU
dc.typeConference Abstracten_AU
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