Abstract
The use of Selenomethionine (SeMet) incorporated protein crystals for single or multiwavelength 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 complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared to native protein expression.
- Abbreviations and symbols
- AAA
- Amino Acid Analysis
- BEVS
- baculovirus expression vector system
- E. coli
- Escherichia coli
- FBS
- Fetal bovine serum
- hCG
- human choriogonadotropin
- Hi5
- Trichoplusia ni
- Leu
- L-leucine
- Met
- L-methionine
- Met+
- L-methionine-containing
- Met-
- L-methionine-free
- MAD
- multi-wavelength anomalous diffraction
- MOI
- multiplicity of infection
- eMOI
- estimated multiplicity of infection
- Phe
- L-phenylalanine
- SAD
- single-wavelength anomalous diffraction
- Sf9
- Spodoptera frugiperda
- Se
- Selenium
- SeMet
- Seleno-L-methionine
- SeAM
- Se-adenosylselenomethionine
- SeAH
- Se-adenosylselenohomocysteine
- SeCys
- Selenocysteine
- S. cerevisiae
- Saccharomyces cerevisiae
- TEQC method
- Titer Estimation for Quality Control method
- Val
- L-valine
Footnotes
Contact Information ytakagi{at}iu.edu
↵2 Present address: Division of Structural Medicine and Anatomy, Kobe University Graduate School of Medicine, Kobe, Hyogo, 650-0017, Japan.