A practical method for efficient and optimal production of Seleno-methionine-labeled recombinant protein complexes in the insect cells.
Sabine WenzelTsuyoshi ImasakiYuichiro TakagiPublished in: Protein science : a publication of the Protein Society (2019)
The use of Seleno-methionine (SeMet) incorporated protein crystals for single or multi-wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared with native protein expression.
Keyphrases
- induced apoptosis
- cell cycle arrest
- escherichia coli
- amino acid
- protein protein
- cell death
- small molecule
- magnetic resonance imaging
- high resolution
- mass spectrometry
- pseudomonas aeruginosa
- cell free
- multidrug resistant
- staphylococcus aureus
- zika virus
- molecular dynamics simulations
- candida albicans
- biofilm formation