Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Induction of specific immunological unresponsiveness by oral autoantigens such as glutamic acid decarboxylase 65 (GAD65) is termed oral tolerance and may be a potential therapy for autoimmune diabetes. However, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. Mucosal adjuvants such as cholera toxin B subunit (CTB) may lower the level of autoantigens required. Here we describe cloning, expression, purification and identification study of the CTB and triple GAD_531–545 epitopes fusion gene. The fusion gene was ligated via a flexible hinge tetrapeptide and expressed as a soluble protein in Escherichia coli BL21 (DE3) driven by the T7 promoter. We purified the recombination protein from the cell lysate and obtained approximately 2.5 mg of CTB–GAD_(531–545)3 per liter of culture with greater than 90% purity by a Ni–NTA resin column. The bacteria produced this protein as the pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and GAD65. Further studies revealed that oral administration of bacterial CTB–GAD_(531–545)3 fusion protein showed the prominent reduction in pancreatic islet inflammation in non-obese diabetic mice. The results presented here demonstrate that the bacteria bioreactor is an ideal production system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes.     

谷氨酸脱羧酶研究进展

谷氨酸脱羧酶(glutamic acid decarboxylase,GAD,EC4.1.1.15)在生物体内广泛存在,其催化产物γ-氨基丁酸(γ-aminobutyric acid,GABA)是哺乳动物体内一种重要的抑制性神经递质。在对自身免疫性疾病以及糖尿病研究中,特别是1型糖尿病,GAD、GABA以及谷氨酸脱羧酶抗体(glutamic acid decarboxylase-antibody,GAD-Ab)等的水平作为病理分析、疾病诊断、免疫治疗的重要参数,历来备受研究者关注。本文就GAD及其催化产物GABA的研究进展进行了综述,为更好地研究自身免疫性疾病的发病机理,探索更加有效安全的治疗方法提供参考。     

Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus

Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A + PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24 h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A + B antisense (PR A + B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A + B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A + B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the regulation of the content of TPH, TH and GAD in the rat hypothalamus.     

Drosophila GABAergic systems II. Mutational analysis of chromosomal segment 64AB, a region containing the glutamic acid decarboxylase gene

The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter -aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.     

The structural and functional heterogeneity of glutamic acid decarboxylase: A review

Studies of the GABA-synthetic enzyme glutamate decarboxylase (glutamic acid decarboxylase; GAD; E.C.4.1.1.15) began in 1951 with the work of Roberts and his colleagues. Since then, many investigators have demonstrated the structural and functional heterogeneity of brain GAD. At least part of this heterogeneity derives from the existence of two GAD genes.In honor of the 70th birthday of Dr. Eugene Roberts     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

The effects of adrenalectomy and thermal stress on glutamic acid decarboxylase activity in different regions of the rat brain

Glutamic acid decarboxylase (GAD) enzyme activity was measured in synaptosomes prepared from the hypothalamus, the hippocampus, the striatum and the cerebral cortex of control, adrenalectomized and rat exposed to a thermal stress. Adrenalectomy caused a statistically significant decrease in the enzyme activity in the striatum, while it had no effect in the other three brain areas. On the other hand, exposure to the thermal stress resulted in a dramatic increase of GAD specific activity in all brain areas examined. This thermal stress-induced increase in enzyme activity was observed in both non-operated and adrenalectomized animals, which implies that it is not mediated by glucocorticoids.Abbreviations used GAD glutamic acid decarboxylase - GABA -aminobutyric acid - AET 2-aminoethylisourethonium bromide - ADX adrenalectomized - rpm revolutions per minute     

Identification and functional analysis of truncated human glutamic acid decarboxylase 65

Human brain glutamate decarboxylase 65 (hGAD65) was found to exist as full-length and truncated forms when the glutathione S-transferase-tagged hGAD65 fusion protein was subjected to factor Xa cleavage. The truncated form is produced by cleavage at arginine 69 based on N-terminal amino acid sequence analysis, and has a molecular weight of 58 kD. It is resistant to further factor Xa cleavage or mild trypsin treatment and is more active and more stable than the full-length form. Both the full-length and truncated forms of GAD are also observed in brain preparations in the presence of protease inhibitors. Furthermore, full-length GAD could be converted to the truncated form by endogenous proteases, suggesting that the conversion of full-length to truncated GAD mediated by endogenous protease may represent an important mechanism in the regulation of GABA biosynthesis in the brain.     

Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.     

Recombinant expression and purification of Ssa1p (Hsp70) from Saccharomyces cerevisiae using Pichia pastoris

Heat shock proteins with a molecular mass of 70000 (Hsp70s) are a ubiquitous class of ATP-dependent molecular chaperones involved in the folding of cellular proteins. Sequencing the entire genome of Saccharomyces cerevisiae revealed 14 different genes for Hsp70 proteins in different cellular compartments. Among these 14 Hsp70s, the subclass of Ssa (Ssa1p-Ssa4p) is abundant and essential in the cytosol. Since high yield expression of cytoplasmic Ssa1p is inefficient in Saccharomyces cerevisiae and recombinant expression in E. coli yields low protein levels, we chose Pichia pastoris as the recombinant expression system. In Pichia pastoris, expression levels of Ssa1p are high and Ssa1p is soluble and correctly folded. Also, we present a new protocol for purification of Ssa1p. Previously described purifications include ATP-agarose chromatography leading to Ssa1p partially complexed with ATP. Our optimized purification protocol follows the CiPP strategy (capture, intermediate purification, polishing) avoiding ATP-agarose chromatography, which allows detailed studies on the ATP-dependent Hsp70 functions. We obtained Ssa1p in high purity and 400 times higher quantity compared to previous studies.     

The ubiquitous 38 kDa contaminant in glutamic acid decarboxylase preparation from the cytosol of Pichia pastoris after immobilised metal ion affinity chromatography is an alcohol dehydrogenase

We expressed a recombinant human glutamic acid decarboxylase (rhGAD) tagged with a hexa-histidine sequence in the Pichia pastoris cytosol. When rhGAD was purified from cell lysates by immobilised metal affinity chromatography, a 38 kDa contaminant protein was evident. This ubiquitous 38 kDa protein was as a yeast alcohol dehydrogenase isozyme that can bind strongly to nickel. Strategies for its removal are discussed.     

Inverse polymerase chain reaction mediated chromosome walking within the human glutamic acid decarboxylase gene

Using inverse polymerase chain reaction (PCR), we have cloned partial intronic sequences from human glutamic acid decarboxylase (GAD) gene. A small 153 bp core region was selected from the GAD cDNA sequence to design outward primers corresponding to its 3′ and 5′ ends. EcoRI digested human DNA which had been circularized by self-ligation and then linearized withSacII was used as a substrate to can.y out PCR. This gave a 900 bp long product which was cloned into pUC19. The sequence analysis of this fragment revealed the presence of introns in the region flanking the selected core DNA. In this work we used this technique to walk into the upsteam region of the GAD gene using sequence information from its cloned cDNA.     

Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris

Caseinomacropeptide is a polypeptide of 64 amino acid residues (106–169) derived from the C-terminal part of the mammalian milk k-casein. This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound. The gene encoding the human caseinomacropeptide (hCMP) was synthesized and expressed with an α-factor secretion signal in the two yeast strains, Saccharomyces cerevisiae and Pichia pastoris. The complete polypeptide of the recombinant hCMP was produced and secreted in a culture medium by both the strains, but the highest production was observed in S. cerevisiae with a galactose-inducible promoter. In a fed-batch bioreactor culture, 2.5 g/l of the recombinant hCMP was obtained from the S. cerevisiae at 97 h.     

Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris

Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml^?1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein ^?1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The K_m and V_max of the GlcN-6-P synthase towards d-fructose 6-phosphate were 2.8 mM and 6.9 μmol min^?1 mg^?1, respectively.     

Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris

As a novel attempt for the intracellular recombinant protein over expression and easy purification from Pichia pastoris, the therapeutic cytokine human granulocyte macrophage colony stimulating factor (hGMCSF) gene was fused to an intein-chitin-binding domain (gene from pTYB11 vector) fusion tag by overlap extension PCR and inserted into pPICZB vector, allowing for the purification of a native recombinant protein without the need for enzymatic cleavage. The fusion protein under the AOX1 promoter was integrated into the P. pastoris genome (SMD 1168) and the recombinant Pichia clones were screened for multicopy integrants. Expression of hGMCSF was done using glycerol and methanol based synthetic medium by three stage cultivation in a bioreactor. Purification of the expressed hGMCSF fusion protein was done after cell disruption and binding of the solubilized fusion protein to chitin affinity column, followed by DTT induced on column cleavage of hGMCSF from the intein tag. In this study, final biomass of 89 g dry cell weight/l and purified hGMCSF of 120 mg/l having a specific activity of 0.657 x 10(7) IU/mg was obtained. This strategy has an edge over the other--His or--GST based fusion protein purification where non-specific protein binding, expensive enzymatic cleavage and further purification of the enzyme is required. It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step.     

林生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究

以林生山黧豆为材料,利用硫酸按分段盐析,丙酮沉淀,DEAE-SepharoseFF离子交换柱层析,SephacrylS300凝胶过滤柱层析及FPLC-MonoQ柱层析技术,以聚酰胺薄膜层析荧光定量法为酶活力检测手段,分离纯化了谷氨酸脱羧酶,达到电泳银染纯．纯化后的林生山黧豆谷氨酸脱羧酶活力达375．09U·mp-1,纯化倍数38．2倍,经SDS-PAGE测定,其亚基分子量为70kD,经梯度PAGE确定,天然分子量为140kD,表明该酶是由两个亚基组成的二聚体．酶学研究表明,纯化的林生山黧豆谷氨酸脱羧酶的最适pH值为5．4,对谷氨酸的Km值为1．62×10-3mol·L-1,酶的最适温度为40℃,酶特异性地使谷氨酸脱羧,不能使天门冬氨酸等其它氨基酸脱羧．     

Characterization and immobilization on nickel-chelated Sepharose of a glutamate decarboxylase A from Lactobacillus brevis BH2 and its application for production of GABA

A gene encoding glutamate decarboxylase A (GadA) from Lactobacillus brevis BH2 was expressed in a His-tagged form in Escherichia coli cells, and recombinant protein exists as a homodimer consisting of identical subunits of 53?kDa. GadA was absolutely dependent on the ammonium sulfate concentration for catalytic activity and secondary structure formation. GadA was immobilized on the metal affinity resin with an immobilization yield of 95.8%. The pH optima of the immobilized enzyme were identical with those of the free enzyme. However, the optimum temperature for immobilized enzyme was 5?°C higher than that for the free enzyme. The immobilized GadA retained its relative activity of 41% after 30 reuses of reaction within 30?days and exhibited a half-life of 19 cycles within 19?days. A packed-bed bioreactor with immobilized GadA showed a maximum yield of 97.8% GABA from 50?mM l-glutamate in a flow-through system under conditions of pH 4.0 and 55?°C.     

Simple purification of aromatic -amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic -amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an M_r of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated -3,4-dihydroxyphenyl-alanine, -5-hydroxytryptophan, and -threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.