TY  - JOUR
TI  - Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
AU  - Yang, Hanting 
AU  - Desai, Nirupa 
VL  - 11
IS  - 15
PY  - 2021
DA  - 2021/08/05
SP  - e4110
C1  - Bio-protocol 2021;11:e4110
DO  - 10.21769/BioProtoc.4110
UR  - https://doi.org/10.21769/BioProtoc.4110
AB  - Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells have been developed. However, the fast purification of mitoribosomes anchored to the mitochondrial inner membrane in complex with the Oxa1L translocase remains particularly challenging. Herein, we present a protocol recently developed and modified in our lab that provides details for the efficient isolation of intact mitoribosomes with its translocase Oxa1L. We combined the cell culture of PDE12-/- or wild-type HEK293 cell lines with the isolation of mitochondria and the purification steps used for the biochemical and structural studies of mitoribosomes and Oxa1L.Graphic abstract:Schematic procedure for the purification of mitoribosomes from HEK cells. The protocol described herein includes two main sections: 1) isolation of mitochondria from HEK cells; and 2) purification of mitoribosome-Oxa1L from mitochondria. RB: Resuspension Buffer (see Recipes) (Created with BioRender.com).
KW  - Mitochondria
KW  - Ribosome purification
KW  - Mitoribosome
KW  - Oxa1L
KW  - Biochemistry
KW  - Cryo-EM
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False