A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

Wenzel, Sabine; Imasaki, Tsuyoshi; Takagi, Yuichiro

doi:10.1002/pro.3575

Title: A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

Journal Article · 04 February 2019 · Protein Science

DOI: https://doi.org/10.1002/pro.3575 · OSTI ID:1493371

Wenzel, Sabine ^[1]; Imasaki, Tsuyoshi ^[1];

^[1]

Department of Biochemistry and Molecular Biology Indiana University School of Medicine 635 Barnhill Drive, Indianapolis, Indiana 46202

Abstract 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.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: NONE; AC02-06CH11357

OSTI ID:: 1493371

Journal Information:: Protein Science, Journal Name: Protein Science Journal Issue: 4 Vol. 28; ISSN 0961-8368

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United Kingdom

Language:: English

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