Ken-Ichi Sano

Cloning of tropomyosins from lobster (Homarus americanus) striated muscles: fast and slow isoforms may be generated from the same transcript.

Complementary DNAs encoding fibre-type-specific isoforms of tropomyosin (Tm) have been isolated from lobster (Homarus americanus) striated muscle expression libraries made from poly(A)+ RNA purified from deep abdominal (fast-type) and crusher-claw closer (slow-type) muscles. A cDNA of slow-muscle Tm (sTm1), containing a complete open reading frame (ORF) and portions of the 5 prime; and 3 prime untranslated regions (UTRs), encodes a protein of 284 amino acid residues with a predicted mass of 32950, assuming acetylation of the amino terminus. The nucleotide sequence of a fast-muscle tropomyosin (fTm cDNA), which includes the entire ORF and part of the 3 prime UTR, is identical to that of sTm1 cDNA, except in the region encoding amino acid residues 39-80 (equivalent to exon 2 of mammalian and Drosophila muscle tropomyosin genes). The deduced amino acid sequences, which display the heptameric repeats of nonpolar and charged amino acids characteristic of alpha-helical coiled-coils, are highly homologous to tropomyosins from rabbit, Drosophila, and shrimp (57% to 99% identities, depending on species). Northern blot analysis showed that two transcripts (1.1 and 2.1kb) are present in both fibre types. Mass spectrometry indicated that fast muscle contains one major isoform (fTm: 32903), while slow muscle contains two major isoforms (sTm1 and sTm2: 32950 and 32884 respectively). Both Tm preparations contained minor species with a mass of about 32830. Sequences of peptides derived from purified slow and fast Tms were identical to the deduced amino acid sequences of the sTm1 and fTm cDNAs, respectively, except in the C-terminal region of fTm. The difference in mass between that predicted by the deduced sequence (32880) and that measured by mass spectrometry (32903) suggests that fTm is post-translationally modified, in addition to acetylation of the N-terminal methionine. These data are consistent with the hypothesis that the fTm and sTm1 are generated by alternative splicing of two mutually-exclusive exons near the 5 prime end of the same gene.

Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus‐infected insect cells. This 21 bp sequence element is derived from a 5′ untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20‐ and seven‐fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A‐rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus‐based expression system.

Autonomous Silica Encapsulation and Sustained Release of Anticancer Protein

We present a novel method for preparing a silica carrier for the sustained release of a proteinaceous pharmaceutical. This method makes use of the silicification activity of the protein itself, which autonomously formed a protein-silica composite upon simple incubation with a silica precursor. The composite was dissolved, and the encapsulated protein was released into a culture medium, thereby sustaining the proteins activity for a long period of time.

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 P43(1)212 with cell constants of . The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of α‐helical coiled‐coil. Thus in the region of 10‐5.5 Å resolution along the , 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 Å along the , whereas along the reflections were limited to 6.6 Å. Data analysis is under way on a data set from a PtCl4 derivative.