Molecular cloning and expression of the sea urchin dynein beta-heavy chain

Ren, Hening
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The DNA sequence that encodes sea urchin dynein β-heavy chain (β-HC) was cloned into a plasmid vector. Plasmid pD1. 3 contains a single 13,168 bp cDNA insert that encodes 98% of the β-HC. The identity of the cDNA insert was verified by partial sequencing and restriction digestion, and was found in agreement with that predicted from the published sequence determined by a PCR based method. Polypeptides encoded by the cloned cDNA reacted positively with antibodies raised against native dynein. Truncated heavy chains of up to 300 kDa were expressed in E. coli, and some were purified under denaturing conditions and refolded. Refolding of one of them, designated RBs12 and representing amino acid residues 2,992-3,501 in the β-HC, yielded a stable soluble protein with possible native-like conformation as judged by its circular dichroic spectrum and solubility in water. Connecting the four nucleotide binding sites in the middle of β-HC with the C-terminal portion of the protein, this region contains two hypothetical coiled-coil structures that was proposed to form the short projection on the dynein head. No specific association of RBs12 with in vitro polymerized microtubules was observed, nor did it significantly block the restoration of beat frequency in KCl-extracted sea urchin sperm flagella by native dynein. The nucleotide binding domains of dynein β-HC were identified by analyzing the homology between dynein isoforms. The putative ATP binding/hydrolysis domain and a second NTP binding domain were expressed in E. coli as fusion or non-fusion proteins. Soluble thioredoxin-fusion proteins were obtained using a low level expression vector, accompanied by high level co-expression of E. coli chaperon 70 from another co-transformed plasmid. The soluble thioredoxin-fusion proteins, purified by Ni-chelating chromatography, were prone to precipitate upon exposure to reduced pH, heat or after prolonged storage. Photo-cleavage of fusion protein representing the ATP binding domain was observed in medium containing millimolar concentrations of vanadate upon irradiation at 365 nm, indicating the presence of native-like conformation. It did not significantly affect the motility of reactivated sea urchin sperm, but slightly inhibited the restoration of beat frequency of KCl extracted sperm by native dynein.
Thesis (Ph. D.)--University of Hawaii at Manoa, 1995.
Includes bibliographical references (leaves 145-154).
xiii, 159 leaves, bound ill., photos. 29 cm
Molecular cloning, Sea urchins -- Genetics
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Biomedical Sciences (Biochemistry); no. 3279
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