Matsujiro Ishibashi

NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt

Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.

Structure of a halophilic nucleoside diphosphate kinase from Halobacterium salinarum

Nucleoside diphosphate kinase from the halophilic archaeon Halobacterium salinarum was crystallized in a free state and a substrate‐bound form with CDP. The structures were solved to a resolution of 2.35 and 2.2 Å, respectively. Crystals with the apo‐form were obtained with His6‐tagged enzyme, whereas the untagged form was used for co‐crystallization with the nucleotide. Crosslinking under different salt and pH conditions revealed a stronger oligomerization tendency for the tagged protein at low and high salt concentrations. The influence of the His6‐tag on the halophilic nature of the enzyme is discussed on the basis of the observed structural properties.

Highly efficient renaturation of β-lactamase isolated from moderately halophilic bacteria

Most, if not all, β‐lactamases reported to date are irreversibly denatured at 60–70°C. Here, we found that a halophilic β‐lactamase from the moderately halophilic bacterium Chromohalobacter sp. 560 was highly stable against heat inactivation: it retained ∼75% of its activity after boiling for 5 min in the presence of 0.2 M NaCl, suggesting that the protein either incompletely denatures during the boiling process or readily renatures upon cooling to the assay temperature. Circular dichroism showed a complete unfolding at 60°C and a full reversibility, indicating that the observed activity after boiling is due to efficient refolding following heat denaturation. The enzyme showed optimal activity at 50–60°C, indicating that an increase in activity with temperature offsets the thermal denaturation. The gene bla was cloned, and the primary structure of the enzyme was deduced to be highly abundant in acidic amino acid residues, one of the characteristics of halophilic proteins. Despite its halophilic nature, the enzyme refolds in low salt media after heat denaturation.

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis.

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis, was achieved by using more stable vectors in the order of S11 replication origin-containing episomal vector < full-length K. lactis plasmid pKD1-containing vector < centromeric vector < chromosome-integrated vectors. Cells containing a PGK (phosphoglycerate kinase) promoter-driven integration vector grown in non-selective rich medium achieved the highest level of secretion, ∼100 μg lysozyme secretion ml −1 culture: this level was ∼10-fold higher than that achieved by episomal vectors. An additional copy of the protein disulfide isomerase gene further facilitated the secretion.

Efficient expression, purification and characterization of mouse salivary α-amylase secreted from methylotrophic yeast, Pichia pastoris

We constructed a secretion vector of mouse salivary α‐amylase, pPAM, using the AOX1 promoter‐terminator and the secretion signal of 128 kDa pGKL killer protein, for an alternative yeast, Pichia pastoris. Taking advantage of multicopy insertion of the expression cassette and optimized growth conditions, we succeeded in highly efficient extracellular production (∼240 µg/ml) of mouse α‐amylase in the 10 ml scale by conventional flask culture: this efficiency was about 90‐fold higher than that of Saccharomyces cerevisiae. Growth temperature of cells was critical for efficient production of α‐amylase. P. pastoris transformants secreted both core‐glycosylated and non‐glycosylated α‐amylase molecules with a glycosylated:non‐glycosylated ratio of about 20:80. Both glycosylated and non‐glycosylated α‐amylases were purified separately to apparent homogeneity. The signal sequence was correctly processed in both species, and the molecular masses of glycosylated and non‐glycosylated α‐amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry. We further studied the outer chain glycosylation of engineered mouse α‐amylase secreted by P. pastoris. Copyright

SECRETION OF MOUSE ALPHA -AMYLASE FROM KLUYVEROMYCES LACTIS

We constructed two mouse α‐amylase secretion vectors for Kluyveromyces lactis using the well‐characterized signal sequence of the pGKL 128 kDa killer precursor protein. Both PHO5 and PGK expression cassettes from Saccharomyces cerevisiae directed the expression of mouse α‐amylase in YPD medium at a similar level of efficiency. K. lactis transformants secreted glycosylated and non‐glycosylated α‐amylase into the culture medium and both species were enzymatically active. The K. lactis/S. cerevisiae shuttle secretion vector pMI6 was constructed, and K. lactis MD2/1(pMI6) secreted about four‐fold more α‐amylase than S. cerevisiae YNN27 harboring the same plasmid, indicating that K. lactis is an efficient host cell for the secretion and production of recombinant proteins.

Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer–dimer interaction and prevent the formation of the tetramer.

Characterization of Arginine as a Solvent Additive: A Halophilic Enzyme Model Protein

Arginine suppresses the aggregation of proteins. However, little is known about its mechanism. Here we have used HsNDK (Halobacterium salinarum nucleoside diphosphate kinase) to examine the solvent property of arginine. After exposure to 2 M arginine, HsNDK was diluted to a low salt buffer, resulting in fully active protein. Since unfolded HsNDK cannot refold in such low salt buffer, the observed activity indicates that HsNDK was in the native state in 2 M arginine. Enzyme activity was also examined directly in the presence of arginine, showing that it was active in the presence of 1 M arginine and, to less extent, 2 M arginine. Arginine, however, could not support refolding of heat-denatured HsNDK. HsNDK was stable at 40 degrees C for 19 h incubation in the presence of 1M arginine.

Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system.

Anti-IZUMO1PFF VHH (variable domain of camelid heavy chain antibody) clones, N6 and N15, from immunized alpaca (Lama pacos) phage library were efficiently expressed and their VHH products were secreted into the culture medium of Brevibacillus choshinensis HPD31-SP3, e.g., at a level of 26-95mg in 100ml conventional flask culture. With a 3-L scale fed-batch culture for 65h, the N15 VHH protein with C-terminal His-tag was produced at ∼3g/l culture medium. The N6 and N15 proteins were easily purified to apparent homogeneity by cation exchange and Ni-affinity chromatographies. Both proteins showed specific antigen-binding activity by ELISA and high antigen binding affinity, KD=6.0-8.6nM, by surface plasmon resonance analysis. Size exclusion chromatography-multi-angle laser light scattering analysis revealed that N6 and N15 proteins purified were exclusively monomeric form in phosphate buffered saline. CD spectrum showed beta-sheet rich structure, consistent with a typical antibody structure and also suggested aromatic-aromatic interactions, as indicated by a positive peak at 232nm. Thermal melting analysis of the N15 protein with C-terminal His-tag demonstrated a clear thermal transition with a Tm at 67°C. The heat-denatured sample recovered antigen binding activity upon cooling, indicating a reversible denaturation.

Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production

Brevibacillus choshinensis (Bacillus brevis) is a protein-hyperproducing bacterium with a useful host-vector system for the production of recombinant proteins. Here, we cloned the ccdA-catA (cmacr;cdA āssociated thioredoxin-like tmacr;hiol-disulfide oxidoreductase) locus of B. choshinensis HPD31-S5. CatA protein (molecular weight, 19664) contains a thioredoxin-like motif, Cys-Gly-Pro-Cys. It was successfully expressed in B. choshinensis extracellularly ( approximately 100 microg x ml(-1) culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly ( approximately 350 microg x ml(-1) culture) with an amino-terminal His-tag. Both recombinant proteins showed thiol-disulfide oxidoreductase activity. Incubation of non-native human epidermal growth factor (hEGF) containing incorrect disulfide bonds with B. choshinensis cells secreting CatA protein resulted in the stimulation of the conversion of non-native hEGF to the native form. Furthermore, co-expression of CatA protein with recombinant hEGF in the B. choshinensis production system increased the yield of native hEGF.