{"id":2078,"date":"2021-07-30T11:09:57","date_gmt":"2021-07-30T11:09:57","guid":{"rendered":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/?page_id=2078"},"modified":"2021-07-30T11:24:20","modified_gmt":"2021-07-30T11:24:20","slug":"seleno-methionine-semet-labeling-of-proteins-in-e-coli","status":"publish","type":"page","link":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/protocols\/seleno-methionine-semet-labeling-of-proteins-in-e-coli\/","title":{"rendered":"Seleno-methionine (SeMet) labeling of proteins in E. coli"},"content":{"rendered":"\n<div class=\"vf-grid | vf-grid__col-3\"><div class=\"vf-grid__col--span-2\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<h3 class=\"wp-block-heading\">Before starting:<\/h3>\n\n\n\n<ul class=\"wp-block-list\"><li>Prepare all required reagents and solutions<\/li><\/ul>\n\n\n\n<h3 class=\"wp-block-heading\">Protocol:<\/h3>\n\n\n\n<p>The seleno-methionine labelling of proteins is based on the use of a methionine auxotrophic&nbsp;<em>E. coli<\/em>strain. For vectors containing a T7 promotor we advice to use the <em>E. coli<\/em> strain B834(DE3). Initially cells are grown in minimal medium containing methionine. Before induction the cells are spun down, the pellet is resuspended in minimum medium and the cells are starved before the addition of seleno-L-methionine. Then overexpression of the target protein is induced and it will incorporate seleno-L-methionine. In some cases the addition of all essential amino acids during the initial growth phase (step 2 &amp; 3) improves results.<\/p>\n\n\n\n<p>To test if the strain is really auxotrophic pick one colony from a minimal medium plate containing methionine to inoculate 5 ml Medium A&nbsp;<strong>without<\/strong>&nbsp;methionine. Incubate the culture overnight at 37\u00b0C. With an methionine auxotrophic strain&nbsp;<strong>no<\/strong>&nbsp;growth should be observed.<\/p>\n\n\n\n<ol class=\"wp-block-list\"><li>Transform the vector into an appropriate <em>E. coli<\/em> expression strain and plate out on minimal medium plates; incubate overnight at 37\u00b0C<\/li><li>Pick a colony and use this to inoculate 5 ml of Medium A plus <strong>5 \u00b5l Methionine (50 mg\/ml)<\/strong>; grow overnight at 37\u00b0C<\/li><li>Add the overnight culture to 1 liter of Medium A plus <strong>1 ml Methionine (50 mg\/ml) <\/strong>and grow the culture at the appropriate temperature until the OD<sub>600<\/sub> ~ 1.0<\/li><li>Centrifuge the cell culture for 10 min. at 4000 rpm and 4\u00b0C<\/li><li>Resuspend the cell pellet in 1 liter Medium A (without Methionine) and grow for 4-8 hours at 37\u00b0C<\/li><li>Add <strong>1 ml Seleno-L-Methionine (50 mg\/ml)<\/strong> and grow for an additional 30 min. at 37\u00b0C<\/li><li>Induce expression of the target protein and continue culturing for 2-12 hours<\/li><li>Harvest the cells by centrifugation at 4\u00b0C<\/li><li>When you don\u2019t continue immediately with the protein purification, freeze the cell pellet and store at -20\u00b0C until further usage<\/li><\/ol>\n\n\n\n<p>Comment: the growth rate of the culture and the optimal induction conditions can vary significantly depending on the vector backbone, the construct design and the solubility of the protein of interest<\/p>\n\n\n\n<h4 class=\"wp-block-heading\">Medium A (per liter):<\/h4>\n\n\n\n<ul class=\"wp-block-list\"><li>100 ml M9 medium (10x)<\/li><li>10 ml trace elements solution (100x)<\/li><li>20 ml 20% (w\/v) glucose<\/li><li>1 ml 1M MgSO<sub>4<\/sub><\/li><li>0.3 ml 1M CaCl<sub>2<\/sub><\/li><li>1 ml biotin (1 mg\/ml)<\/li><li>1 ml thiamin (1 mg\/ml)<\/li><li>Appropriate antibiotics<\/li><\/ul>\n\n\n\n<h4 class=\"wp-block-heading\">10x M9 medium (per liter)<\/h4>\n\n\n\n<ul class=\"wp-block-list\"><li>60 g Na<sub>2<\/sub>HPO<sub>4<\/sub><\/li><li>30 g KH<sub>2<\/sub>PO<sub>4<\/sub><\/li><li>5 g NaCl<\/li><li>5 g NH<sub>4<\/sub>Cl<\/li><\/ul>\n\n\n\n<h4 class=\"wp-block-heading\">100 x trace elements solution (per liter):<\/h4>\n\n\n\n<ul class=\"wp-block-list\"><li>5 g EDTA<\/li><li>0.83 g FeCl<sub>3<\/sub>.6H<sub>2<\/sub>O<\/li><li>84 mg ZnCl<sub>2<\/sub><\/li><li>13 mg CuCl<sub>2<\/sub>.2H<sub>2<\/sub>O<\/li><li>10 mg CoCl<sub>2<\/sub>.6H<sub>2<\/sub>O<\/li><li>10 mg H<sub>3<\/sub>BO<sub>3<\/sub><\/li><li>1.6 mg MnCl<sub>2<\/sub>.6H<sub>2<\/sub>O<\/li><\/ul>\n\n\n\n<p>Comment: First dissolve the 5 g EDTA in 800 ml water and adjust the pH to 7.5. Then add the other components and adjust the volume to 1 L. Sterilize the solution by filtration through a 0.22 \u00b5m filter.<\/p>\n\n\n\n<h4 class=\"wp-block-heading\">Stock solutions<\/h4>\n\n\n\n<ul class=\"wp-block-list\"><li>20% (w\/v) glucose (sterilized)<\/li><li>1M MgSO<sub>4 <\/sub>(sterilized)<\/li><li>1M CaCl<sub>2<\/sub> (sterilized)<\/li><li>1 mg\/ml Biotin (filter sterilized)<\/li><li>1 mg\/ml Thiamin (filter sterilized)<\/li><li>50 mg\/ml Methionine (filter sterilized)<\/li><li>50 mg\/ml Seleno-L-Methionine (filter sterilized)<\/li><\/ul>\n\n<\/div>\n<\/div>\n\n\n<div><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":0,"parent":2060,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"template-title-left-aligned.php","meta":{"_acf_changed":false,"footnotes":""},"embl_taxonomy":[],"class_list":["post-2078","page","type-page","status-publish","hentry"],"acf":[],"embl_taxonomy_terms":[],"_links":{"self":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2078","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/comments?post=2078"}],"version-history":[{"count":3,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2078\/revisions"}],"predecessor-version":[{"id":2124,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2078\/revisions\/2124"}],"up":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2060"}],"wp:attachment":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/media?parent=2078"}],"wp:term":[{"taxonomy":"embl_taxonomy","embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/embl_taxonomy?post=2078"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}