Overexpression of the 77-kD echinoderm microtubule-associated protein (EMAP), a WD-40 repeat protein, in baculovirus-infected Sf9 cells

Daggett, Melissa A. F.; Li, Qingwen; Weaver, Robert F.; Suprenant, Kathy A.

doi:10.1002/(sici)1097-0169(1998)41:1<57::aid-cm5>3.0.co;2-c

Cell Motil. Cytoskeleton

1998

DOI: 10.1002/(sici)1097-0169(1998)41:1<57::aid-cm5>3.0.co;2-c

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Overexpression of the 77-kD echinoderm microtubule-associated protein (EMAP), a WD-40 repeat protein, in baculovirus-infected Sf9 cells

Melissa A. F. Daggett

Qingwen Li

Robert F. Weaver

et al.

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Cited by 2 publications

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Saturable binding of the echinoderm microtubule‐associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments

Eichenmüller

Ahrens

et al. 2001

Cell Motil. Cytoskeleton

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The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin.

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Saturable binding of the echinoderm microtubule‐associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments

Eichenmüller

Ahrens

et al. 2001

Cell Motil. Cytoskeleton

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Human EML4, a novel member of the EMAP family, is essential for microtubule formation

Pollmann¹,

Parwaresch²,

Adam‐Klages

et al. 2006

Experimental Cell Research

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Overexpression of the 77-kD echinoderm microtubule-associated protein (EMAP), a WD-40 repeat protein, in baculovirus-infected Sf9 cells

Cited by 2 publications

References 56 publications

Saturable binding of the echinoderm microtubule‐associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments

Saturable binding of the echinoderm microtubule‐associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments

Human EML4, a novel member of the EMAP family, is essential for microtubule formation

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