Expression of chick and yeast β-tubulin-encoding genes in insect cells

Vats-Mehta, Sushma; Yarbrough, Lynwood R.

doi:10.1016/0378-1119(93)90572-k

Cited by 3 publications

(1 citation statement)

References 24 publications

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“…Expression of heterologous tubulins has so far been reported only for mammalian [12]and insect cells [13]. We here describe the first successful expression of foreign tubulins in yeast cells as well as the first coexpression of protein subunits in the P. pastoris system.…”

Section: Introductionmentioning

confidence: 99%

Expression of Reticulomyxa filosa tubulins in Pichia pastoris: regulation of tubulin pools

Linder¹,

Schliwa²,

Kube-Granderath³

1997

FEBS Letters

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We expressed the oc2-and ß2-tubulin isoforms of the giant freshwater amoeba Reticulomyxa filosa in the methylotrophic yeast Pichia pastoris. Single expression lead to little or no detectable material. Coexpression of both tubulins, however, resulted in a significant increase of expressed proteins. At the same time, the detectable internal tubulins of the host yeast cell were downregulated. This finding indicates the functionality of the expressed amoeba tubulins. Further regulation phenomena were observed on the level of equilibrium between the two R. filosa tubulin isoforms and on the level of the total tubulin pool. The P. pastorislR. filosa system therefore seems to be an accessible system for the simultaneous study of the various mechanisms involved in tubulin regulation.

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Section: Introductionmentioning

confidence: 99%

Expression of Reticulomyxa filosa tubulins in Pichia pastoris: regulation of tubulin pools

Linder¹,

Schliwa²,

Kube-Granderath³

1997

FEBS Letters

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Tubulin Genes and Promotors

Breviario

2000

Plant Microtubules

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Identification of intrinsic dimer and overexpressed monomeric forms of γ-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas γ-tubulin sequence

Vassilev

Kimble

Silflow

et al. 1995

Journal of Cell Science

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A new member of the tubulin superfamily, gamma-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa gamma-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell supernatant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin separated into two major peaks. The lagging peak contained a monomeric form of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. The released gamma-tubulin monomers were capable of binding to microtubules in vitro and biochemical quantities of active monomers were further purified using a combination of size-exclusion and ion-exchange column chromatography. The endogenous Sf9 cell gamma-tubulin migrated faster than Chlamydomonas gamma-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas gamma-tubulin was present in a monomeric form, endogenous gamma-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).

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Expression of chick and yeast β-tubulin-encoding genes in insect cells

Cited by 3 publications

References 24 publications

Expression of Reticulomyxa filosa tubulins in Pichia pastoris: regulation of tubulin pools

Expression of Reticulomyxa filosa tubulins in Pichia pastoris: regulation of tubulin pools

Tubulin Genes and Promotors

Identification of intrinsic dimer and overexpressed monomeric forms of γ-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas γ-tubulin sequence

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