Potential roles of recombinant acetylcholine receptor α subunit 1-211 in immunoadsorbent and DNA immunization.

The open reading frame of the α-subunit (amino acids 1-211) of human muscle nicotinic acetylcholine receptor (hAChR(α211)) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR(α211) (recombinant hAChR(α211)) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K-hAChR(α211) with a yield of 25 mg/L. rhAChR(α211) was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChR(α211) was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA-hAChR(α211). Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patient's sera and establish an animal model of myasthenia gravis by recombinant hAChR(α211) immunization.

Quantitative analysis and functional evaluation of zinc ion in the D-hydantoinase from Pseudomonas putida YZ-26.

D-hydantoinase (HDT) is a metal-dependent enzyme that is widely used in industrial bioconversion to D-amino acids as valuable intermediates in the fields of food, pharmaceutical industry and agriculture. In this report, we prepared apo-HDT (metal-removed HDT) and Zn(2+)-HDT (Zn(2+)-added HDT) in vitro from a recombinant HDT (re-HDT) expressed in E. coli. The Zn(2+)-HDT and re-HDT contain 2.17 and 0.95 mol Zn(2+) per mol subunit, respectively, and they have comparable enzymatic activities. In contrast, the apo-HDT only retains 0.04 mol Zn(2+) per mol subunit with less than 10% activity, compared with the re-HDT. When the apo-HDT was reconstituted with ZnCl(2), the enzymatic activity recovery was about 75%. Moreover, the fluorescence intensity, circular dichroism spectra and thermo-stability of the apo-HDT and Zn(2+)-HDT are quite different from those of the re-HDT. These data suggest that the re-HDT may have two Zn(2+)-binding sites, one is an intrinsic or tight-binding site (zinc-alpha) essential for its activity and the other is a vacant or loose-binding site (zinc-beta) possibly non-essential for the activity.

Over-expression, rapid preparation and some properties of c-terminal BARc region in PICK1.

A DNA fragment encoding C-terminal BARc region (amino acids 128-416) of rat PICK1 (NP_445912 ) was inserted into a modified vector pMAL-s involving human rhinovirus 3C protease cleavage site to produce a recombinant plasmid, pMAL-s-barc. The construct can express the fusion protein, MBP-BARc in the soluble form in E.coli. To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE. The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate. In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination.

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.

Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain.

PICK1 (protein interacting with Ckinase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn2+-binding site. The Zn2+-binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C-terminal acidic region). Deleting the N-terminal acidic region of NPDZ domain (involving PDZ domain and N-terminal acidic region) in PICK1 impairs its Zn2+-binding capacity. The mutation of the CPC (Cys-Pro-Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn2+-binding. In addition, Zn2+ can enhance the lipid-binding ability of PDZ domain as observed in both protein-lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn2+ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane.

Expression of a buckwheat trypsin inhibitor gene in Escherichia coli and its effect on multiple myeloma IM-9 cell proliferation.

The gene of buckwheat trypsin inhibitor (BTI) has been cloned and expressed in Escherichia coli. The yield of this recombinant inhibitor was over 12 mg/L by using one-step purification on a Ni2+-NTA Sepharose column. Its molecular weight was 9322.1 Da, determined by mass spectrum analysis. The MTT and cytometry analyses showed that recombinant BTI could specifically inhibit the proliferation of IM-9 human B lymphoblastoid cells (from patient with multiple myeloma) in a dose-dependent manner. The test of recombinant BTI-induced apoptosis in IM-9 cells implied that the inhibitor might have potential application in the treatment of cancer.

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.

Truncations of gelonin lead to a reduction in its cytotoxicity.

Gelonin is a single chain ribosome-inactivating protein (RIP) with potential applications as a bullet of immunoconjugate for the treatment of cancer and AIDS. Using truncated forms of gelonin, we now report the relationship between its conformation and function. Circular dichroism (CD) and fluorescence spectra show that the N-terminus forms beta-sheets whereas the C-terminus contains alpha-helices of secondary structures. Biological experiments indicate that all gelonin truncation mutants lose partial toxicity compared to intact gelonin, an effect most strongly seen with C-terminally truncated gelonin. Similar evidence is also provided using a DNase-like activity assay. In addition, the intact gelonin exhibits the highest cytotoxicity to cancer cells. These results suggest that truncations of the terminal region of gelonin negatively regulate its function dominantly and that, due to its toxicity, intact gelonin is an important potential immunoconjugate.

Subunit dissociation and stability alteration of D hydantoinase deleted at the terminal amino acid residue.

Two variants of D hydantoinase (HYD), created by deletion of one amino acid residue of at either the N- or C-terminus, were expressed in Escherichia coli and purified by two-step chromatography. Compared with HYD, HYDc1 with the C-terminal Arg deletion retained 43% activity, while HYDn1 with the N-terminal Ser deletion had no activity using DL Hydantoin as substrate. Based on HYD dimer with a molecular weight of 103 kDa, HYDc1 is a monomer of 52 kDa and HYDn1 is a mixture of dimer and monomer. Moreover, HYDc1 displayed higher pH stability and lower thermal stability compared to HYD. In addition, the secondary and tertiary structures of HYDc1 were not significantly changed in contrast to the ones of HYDn1. All data imply that the C-terminal Arg of the HYD is crucial for homodimeric architecture of the enzyme, but non-essential for catalysis, while the N-terminal Ser is required for both conformation and catalysis of the enzyme.

Novel immunotoxin: a fusion protein consisting of gelonin and an acetylcholine receptor fragment as a potential immunotherapeutic agent for the treatment of Myasthenia gravis.

In continuation of our attempts for antigen-specific suppression of the immune system [I.L. Urbatsch, R.K.M. Sterz, K. Peper, W.E. Trommer, Eur. J. Immunol. 23(1993) 776-779] a novel fusion protein composed of amino acids 4-181 of the extracellular domain of the alpha-subunit of the human muscle acetylcholine receptor and the plant toxin gelonin was expressed in Escherichia coli. The fusion protein formed inclusion bodies but could be solubilized in the presence of guanidinium hydrochloride. After a simple two step purification and refolding procedure, it exhibited a native structure at least in the main immunogenic region as shown by antibodies recognizing a conformational epitope. Half maximal inhibition of translation was achieved at 46 ng/ml as compared to 4.6 ng/ml for native and 2.4 for recombinant gelonin. Its use as therapeutic agent for the treatment of Myasthenia gravis was investigated in an animal model. Female Lewis rats were immunized with complete acetylcholine receptor from the electric ray Torpedo californica and developed thereafter experimental autoimmune M. gravis. Quantitative assessment of the disease was achieved by repetitive stimulation of the Nervus tibialis. Rats showed no symptoms of M. gravis, neither visually nor electrophysiologically after treatment with the fusion protein as determined one and seven weeks after the second application. This approach may also be useful for the therapy of further autoimmune diseases by substituting other autoantigens for the AchR fragment in the fusion protein.

[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.

Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin.

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.

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).

[Purification and some properties of D-carbamoylase].

A D-Carbamoylase produced by a strain NO. 2262 was purified to electrophoretic homogeneity with the recovery of 20% activity and the purification factor of 8 fold by three steps including (NH4)2SO4 fractionation, hydrophobic column and pre-packed Hitrap Q HR. It is indicated from the results of nativ-PAGE and SDS-PAGE analysis that the enzyme could be a homogeneous tetramer consisting of four 35 kD subunits. In addition, its optimal pH and optimal temperature are 8.0 and 45 degrees C respectively. The basic kinetic parameters of the enzyme are Km = 1.3 x 10(-3) mol/L and Vmax = 0.33 mumol/min with N-carbamyl-DL-Alanine as the substrate. The effect of bivalent metal ions on the enzyme was showed that Ni2+ could be as an activator, Zn2+ as a powerful inhibitor, while Co2+ had no any influence at all. Its N-terminal sequence is TRQKILAF in turn.