DOI: 10.1101/491548Dec 9, 2018Paper

A practical method for efficient and optimal production of selenomethionine-labeled recombinant protein complexes in the insect cells

BioRxiv : the Preprint Server for Biology
Sabine WenzelYuichiro Takagi

Abstract

The use of Selenomethionine (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 E. 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 allows for incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein co...Continue Reading

Related Concepts

Separation Anxiety Disorder
Biological Markers
Escherichia coli
Insecta
Recombinant Proteins
Selenomethionine
X-Ray Diffraction
Crystallography, X-Ray
Crystal Structure
Toxic Effect

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