MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Overexpression, purification, and functional analysis of recombinant human tubulin dimer Overexpression, purification, and functional analysis of recombinant human tubulin dimer FEBS Lett. (0014-5793) 2013 imex curation Minoura I., Hachikubo Y., Yamakita Y., Takazaki H., Ayukawa R., Uchimura S., Muto E. emuto2@brain.riken.jp 30-Sept-2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro affinity chrom affinity chromatography technology Affinity purification Affinity purification affinity chrom de novo protein sequence de novo protein sequencing by mass spectrometry de novo protein sequence Minoura I., Hachikubo Y., Yamakita Y., Takazaki H., Ayukawa R., Uchimura S., Muto E. emuto2@brain.riken.jp Accepted 2013-AUG-29 by LPERFETTO 30-Sept-2013: Contacted by IntAct-Help. FEBS Lett. (0014-5793) 2013 Only protein-protein interactions imex curation Overexpression, purification, and functional analysis of recombinant human tubulin dimer Overexpression, purification, and functional analysis of recombinant human tubulin dimer FEBS Lett. (0014-5793) 2013 imex curation Minoura I., Hachikubo Y., Yamakita Y., Takazaki H., Ayukawa R., Uchimura S., Muto E. emuto2@brain.riken.jp 30-Sept-2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro light microscopy light microscopy predetermined predetermined participant predetermined Minoura I., Hachikubo Y., Yamakita Y., Takazaki H., Ayukawa R., Uchimura S., Muto E. emuto2@brain.riken.jp Accepted 2013-AUG-29 by LPERFETTO 30-Sept-2013: Contacted by IntAct-Help. FEBS Lett. (0014-5793) 2013 Only protein-protein interactions imex curation Overexpression, purification, and functional analysis of recombinant human tubulin dimer Overexpression, purification, and functional analysis of recombinant human tubulin dimer FEBS Lett. (0014-5793) 2013 imex curation Minoura I., Hachikubo Y., Yamakita Y., Takazaki H., Ayukawa R., Uchimura S., Muto E. emuto2@brain.riken.jp 30-Sept-2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro light scattering light scattering predetermined predetermined participant predetermined Minoura I., Hachikubo Y., Yamakita Y., Takazaki H., Ayukawa R., Uchimura S., Muto E. emuto2@brain.riken.jp Accepted 2013-AUG-29 by LPERFETTO 30-Sept-2013: Contacted by IntAct-Help. FEBS Lett. (0014-5793) 2013 Only protein-protein interactions imex curation tba1b_human Tubulin alpha-1B chain TUBA1B Tubulin alpha-ubiquitous chain Alpha-tubulin ubiquitous Tubulin K-alpha-1 protein protein human Homo sapiens Human MRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGKHVPRAVFVDLEPTVIDEVRTGTYRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLVLDRIRKLADQCTGLQGFLVFHSFGGGTGSGFTSLLMERLSVDYGKKSKLEFSIYPAPQVSTAVVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLISQIVSSITASLRFDGALNVDLTEFQTNLVPYPRIHFPLATYAPVISAEKAYHEQLSVAEITNACFEPANQMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTKRSIQFVDWCPTGFKVGINYQPPTVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYVGEGMEEGEFSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEY 94355B4EC2086429 tbb3_human Tubulin beta-3 chain TUBB3 TUBB4 Tubulin beta-III Tubulin beta-4 chain protein protein human Homo sapiens Human MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPSGNYVGDSDLQLERISVYYNEASSHKYVPRAILVDLEPGTMDSVRSGAFGHLFRPDNFIFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKECENCDCLQGFQLTHSLGGGTGSGMGTLLISKVREEYPDRIMNTFSVVPSPKVSDTVVEPYNATLSIHQLVENTDETYCIDNEALYDICFRTLKLATPTYGDLNHLVSATMSGVTTSLRFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTARGSQQYRALTVPELTQQMFDAKNMMAACDPRHGRYLTVATVFRGRMSMKEVDEQMLAIQSKNSSYFVEWIPNNVKVAVCDIPPRGLKMSSTFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATAEEEGEMYEDDEEESEAQGPK DIP protein Q13509 original sequence version: 137 4B9CDE7DBA102949 tuba1b-tubb3 1 alpha1-tubulin 4 unspecified role unspecified role unspecified role unspecified role partially purified partially purified tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. beta3-tubulin 5 unspecified role unspecified role unspecified role unspecified role partially purified partially purified tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. association association f2 f3b f3c The presence of both endogenous and recombinant alpha-tubulin in the eluent from the His-affinity column indicates that His-tagged alpha1-tubulin can form dimers with either type of alpha-tubulin (Fig. 2D). Vice versa, when DEAE eluent was first applied to the FLAG-affinity column instead of the Hisaffinity column, the eluent from the FLAG-affinity column contained both endogenous and recombinant alpha±-tubulin, indicating that alpha3-tubulin can form dimers with either type of alpha±-tubulin (data not shown). Because recombinant tubulin forms dimers with its endogenous counterpart, certain fractions of soluble alpha1- and alpha3-polypeptides originally expressed in the HighFive cells (Fig. 1B) were inevitably lost during the purification process (Fig. 2D). tuba1b-tubb3-1 3 beta3-tubulin 5 unspecified role unspecified role unspecified role unspecified role partially purified partially purified tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. alpha1-tubulin 4 unspecified role unspecified role unspecified role unspecified role partially purified partially purified tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. physical association physical association f4a Tubulin assembled in the presence of 2 M taxol for 30 min was further stabilized by increasing the taxol concentration to 50 M and was examined under a dark-field microscope. The recombinant microtubules appeared uniform, like those isolated from pig brain (Fig. 4B), but with a greater length than that of brain microtubules (16.2 +/- 11.9 (N = 72, mean SD) and 7.9 +/- 10.2 m (N = 81) for recombinant and brain microtubules, respectively, Fig. 4C). tuba1b-tubb3-2 2 beta3-tubulin 5 unspecified role unspecified role unspecified role unspecified role partially purified partially purified tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. alpha1-tubulin 4 unspecified role unspecified role unspecified role unspecified role partially purified partially purified tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. physical association physical association f4b To measure the assembly kinetics, purified K40R tubulin (0.2 mg/mL) was mixed with 2 M taxol, and the process of polymerization was monitored by 350-nm light scattering. The result shows slower polymerization of recombinant tubulin compared to pig brain tubulin (Fig. 4A).