RESUMO
We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of 2H, 13C and 15N using auto-induction or isopropyl-ß-D-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.
Assuntos
Marcação por Isótopo/economia , Ressonância Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono , Deutério , Escherichia coli/metabolismo , Fermentação , Marcação por Isótopo/métodos , Metionina/análogos & derivados , Isótopos de Nitrogênio , Reprodutibilidade dos TestesRESUMO
We describe an improved, universal method for the seamless integration of DNA fragments into plasmids at any desired position. The protocol allows in vitro joining of insert and linearized plasmid at terminal homology regions using the BD In-Fusion cloning system. According to the standard BD In-Fusion protocol, vectors are linearized by restriction enzyme digestion. Linearization of plasmids by polymerase chain reaction (PCR), instead of restriction enzyme digestion, extends the usefulness of the method by rendering it independent of restriction endonuclease recognition sites and by allowing seamless insertion of DNA fragments at any position, without introduction of unwanted nucleotides flanking the site of insertion. The combination of PCR linearization of plasmids and BD In-Fusion technology has shown to be very useful for the insertion of genes into the expression regions of multiple plasmids for the heterologous expression of proteins in Escherichia coli. Hands-on time is minimal and there is no need for preparative gel electrophoresis. The protocol is very simple and only involves PCR and liquid handling steps. The method should therefore theoretically have a good potential for automation.
