Molecular cloning, recombinant expression, and immunological characterization of a novel allergen from tartary buckwheat.

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.

The flexibility of the non-conservative region at the C terminus of D-hydantoinase from Pseudomonas putida YZ-26 is extremely limited.

We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of D-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

Gene cloning, expression, and substrate specificity of an imidase from the strain Pseudomonas putida YZ-26.

A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni(2+)-NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.

Induction of apoptosis by buckwheat trypsin inhibitor in chronic myeloid leukemia K562 cells.

Buckwheat is an ancient and specialty grain in China. Due to its unique chemical and bio-activity components, buckwheat has been found to have many uses in food products and medicine. However, very little is known about the toxicity of protease inhibitors from buckwheat. Here, the possible effects of a recombinant buckwheat trypsin inhibitor (rBTI) on the induction of apoptosis of the human K562 cell line were investigated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays and flow cytometric analysis. MTT assay showed that rBTI could specifically inhibit the growth of K562 cells in a dose-dependent manner, but there were minimal effects on normal human peripheral blood mononuclear cells (PBMCs). Furthermore, comparison the effects of rBTI on K562 cells with those of negative control (BSA and the complex of BSA and rBTI) revealed that rBTI was highly toxic to K562 cells, and BSA hardly had any inhibition on proliferation in K562 cells. The analysis of flow cytometric indicated that the apoptosis of K562 cells were 31.0%, 32.8%, 35.3% and 52.1% after treated by rBTI in range of 12.5-100 microg/ml, respectively. The results suggested that rBTI can induce apoptosis of K562 cells and that it might be a potential protein drug of the trypsin inhibitor family.

[Molecular form and stability of D-hydantoinase deleted at C-terminal residue Arg].

This report is about only deleting one C-terminal residue of D-hydantoinase to result in obvious changes on its molecular form and stability. A recombinant D-hydantoinase (P479) and its mutant enzyme deleted at C-terminal residue Arg (P478) were prepared by methods of gene cloning, expression and purification. Results show that the subunit molecular weight of P479 and P478 is the same (54kDa) as determined by SDS-PAGE, whilst the molecular form of native P479 and P478 is a dimer and a monomer respectively in the completely operative conditions. Compared with P479, the enzymatic activity of P478 for substrate hydantoin maintained about 40% and pH stability was obviously increased, at the alkaline side in particular, as well as the anti-SDS ability was also raised. However, the thermal stability for P478 was clearly lowed as compared to P479. It implies from above data that the C-terminal residue Arg of the D-hydantoinase is a crucial one for subunit dissociation, but non-essential for catalysis.

Preparation of an immunoadsorbent coupled with a recombinant antigen to remove anti-acetylcholine receptor antibodies in abnormal serum.

An immunoadsorbent that removes anti-acetylcholine receptor antibodies (AChRAb) in abnormal serum of myasthenia gravis (MG) patient was efficiently prepared by an expression product, the functional fragment of AChR(alpha205) fused with maltose binding protein (MBP). The ligand can then covalently bind to amylose resin through MBP fusion protein. It was shown from the result of this study with anti-AChR mice sera that the removal rate of AChRAb on this immunoadsorbent reached 87+/-10% (mean value of 10 mice) and the maximally binding capacity of AChRAb was approximately 260 microg/g immunoadsorbent (wet weight). Moreover, the immunoadsorption test of sera in two MG patients indicated that about 90% and 96% of abnormal AChRAb could be eliminated, while other serum components such as albumin, IgG, IgM and IgA only dropped 18%, 35%, 22%, 15% and 24%, 27%, 15%, 12%, respectively, for two MG patient sera. It is anticipated from this study that the immunoadsorbent reported here could, with further development, find its clinical application for removal of AChRAb from patient serum.

Soluble expression and affinity purification of functional domain of human acetylcholine receptor alpha-subunit by the modulation of maltose binding protein.

An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR alpha205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR alpha205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR alpha205 and AChR alpha205 were similar to that of AChR alpha-subunit from Torpedo.

Influence of inocula and grains on sclerotia biomass and carotenoid yield of Penicillium sp. PT95 during solid-state fermentation.

Various inocula and grains were evaluated for carotenoid production by solid-state fermentation using Penicillium sp. PT95. Millet medium was more effective in both sclerotia growth and carotenoid production than other grain media. An inoculum in the form of sclerotia yielded higher sclerotia biomass compared to either a spore inoculum or a mycelial pellet inoculum. Adding wheat bran to grain medium favored the formation of sclerotia. However, neither the inoculum type nor addition of wheat bran resulted in a significant change in the carotenoid content of sclerotia. Among grain media supplemented with wheat bran (wheat bran:grain =1:4 w/w, dry basis), a medium consisting of rice and wheat bran gave the highest sclerotia biomass (15.10 g/100 g grain), a medium consisting of buckwheat and wheat bran gave the highest content of carotenoid in sclerotia (0.826 mg/g dry sclerotia), and a medium consisting of millet and wheat bran gave the highest carotenoid yield (11.457 mg/100 g grain).