Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells.
A new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin was prepared by removing 11 amino acids at the C-terminus. The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations. By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals. The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart. For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P4(3)(1)2(1)2 with cell constants of a=b=105.6 angstrom, c=506.9 angstrom. The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of alpha-helical coiled-coil. Thus in the region of 10-5.5 angstrom resolut along the c*-axis, the reflections were weak. For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background. There the crystals diffracted to beyond 3.0 A along the c*-axis, whereas along the a*-b*-plane reflections were limited to 6.6 angstrom. Data analysis is under way on a data set from a PtCl4 derivative.
Miegel
A
A
International Institute for Advanced Research, Central Research Laboratories, Seika, Kyoto, Japan.
Sano
K I
KI
Yamamoto
K
K
Maeda
K
K
Maéda
Y
Y
Taniguchi
H
H
Yao
M
M
Wakatsuki
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002460:Cell Line; D003460:Crystallization; D018360:Crystallography, X-Ray; D004121:Dimethyl Sulfoxide; D015870:Gene Expression; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000737:chemistry; D008121:Nephropidae; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis; D017384:Sequence Deletion; D018411:Spodoptera / Q000235:genetics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014335:Tropomyosin; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D004121:Dimethyl Sulfoxide