Purification and characterization of human dehydrodolychil diphosphate synthase (DHDDS) overexpressed in E. coli.
Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrodolichyl diphosphate synthase (DHDDS) is an eukaryotic cis prenyltransferase (cis-PT) that catalyzes chain elongation of farnesyl diphosphate via multiple condensations with isopentenyl diphosphate to form dehydrodolichyl diphosphate, a precursor for the glycosyl carrier dolichylpyrophophate involved in N-linked glycosylation. Mutations in DHDDS were shown to result in retinitis pigmentosa, ultimately leading to blindness, but the exact molecular mechanism by which the mutations affect DHDDS function remains elusive. In addition, bacterial cis-PT homologs are involved in bacterial wall synthesis and are therefore potential targets for new antibacterial agents. However, as eukaryotic cis-PT were not thoroughly characterized structurally and functionally, rational design of prokaryotic cis-PT specific drugs is currently impossible. Here, we present a simple protocol for purification of functionally active human DHDDS under non-denaturating conditions using a codon-optimized construct. The purified protein forms a stable homodimer, similar to its bacterial homologs, and shows time- and substrate-dependent activity. Purification of this protein requires the presence of a detergent for protein solubility. The protocol described here may be utilized for the overexpression of other eukaryotic cis-PT. Future structural and functional studies of the recombinant DHDDS may shed light on the mechanisms underlying DHDDS-related retinitis pigmentosa and lead to novel therapeutic approaches.
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. Electronic address: moshegil@post.tau.ac.il.
Moshe
M
Giladi
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Ilan
I
Edri
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Michal
M
Goldenberg
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Ophthalmology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
Hadas
H
Newman
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Roi
R
Strulovich
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Daniel
D
Khananshvili
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Electronic address: yhaitin@post.tau.ac.il.
Yoni
Y
Haitin
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Ophthalmology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
Anat
A
Loewenstein
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C051389:dehydrodolichyl diphosphate synthetase
DHDDS; Dehydrodolychil diphosphate synthase; Dolichol; Enzyme; Heterologous expression; Prenyltransferase