Application of fed-batch fermentation to the preparation of isotopically labeled or selenomethionyl-labeled proteins.
An increasing demand for isotopically labeled samples for spectroscopic and crystallographic studies has led to a corresponding need for effective and efficient methods for producing these samples. The present work is based on the strategy of using an isotopically labeled compound as the growth-limiting nutrient during protein expression in Escherichia coli (DE3) strains. By using dissolved O2 and agitation rate data, the cell growth, feeding of the isotopic label, induction of protein expression, and the harvest of cells can be coordinated in a feedback controlled fermenter in a simple, easily defined manner. This approach is demonstrated for the nutrient-limited production of [U-15N]- and [U-13C, U-15N]-labeled toluene 4-monooxygenase effector protein in E. coli BL21(DE3) with isotopic abundance identical to that of the labeled precursors. For selective labeling, demonstrated with selenomethionine using methionine auxotroph E. coli B834(DE3), approximately 80-85% incorporation was obtained from methionine-dependent growth of the auxotroph followed by selenomethionine feeding and protein induction upon methionine depletion. This selective labeling is accomplished in a single culture, does not require washing or resuspension, minimizes costly incorporation of label into host cell mass prior to induction, and can be easily adapted to selective labeling with other amino acids. Moreover, cell mass yield from these experiments can be readily optimized to provide the desired level of protein for a given investigation from a single growth and purification. This combination provides an efficient, controllable option for isotopic labeling experiments.
Studts
J M
JM
Graduate School and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Fox
B G
BG
D001483:Base Sequence; D002247:Carbon Isotopes; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005798:Genes, Bacterial; D009587:Nitrogen Isotopes; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D012645:Selenomethionine / Q000378:metabolism
D002247:Carbon Isotopes; D017931:DNA Primers; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D012645:Selenomethionine; D010105:Oxygenases; C068610:toluene-4-monooxygenase