High-level expression of a soluble snake venom enzyme,gloshedobin, in E. coli in the presence of metal ions

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21(DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 °C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg²⁺ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.     

长白山白眉蝮蛇毒类凝血酶Gussurobin基因的克隆、表达与纯化

根据同源性 ,在高度保守的上游信号肽区域设计引物 ,通过RT PCR反应 ,从长白山白眉蝮蛇 (Gloydiusussurensis)毒腺总RNA中克隆得到类凝血酶 gussurobincDNA ,双向测序得到 gussurobin基因的全序列并由此推测出相应的氨基酸序列。与其他已知的类凝血酶不同 ,gussurobin只含有一个可能的糖基化位点 ,即Asn12 4 Ser12 5 Thr12 6。将gussurobin基因克隆到表达载体 pPIC9K中 ,电极转化至毕氏酵母菌株GS115中 ,经G418抗性筛选和营养缺陷型筛选获得重组子。经摇瓶培养 ,获得表达。经过柱层析分离 ,获得SDS PAGE电泳纯的重组gussurobin。     

Expression of gloshedobin,a thrombin-like enzyme from the venom of Gloydius shedaoensis,in Escherichia coli

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, was expressed in Escherichia coli using expression vector pET-32a(+). The gene was expressed under T7 promotor with a fusion partner of Thx.Tag and a 6xHis.Tag at its 5 terminal. After induction by IPTG for 6 h, the recombinant enzyme was expressed in the cytoplasm. Expression at 25°C gave twice the amount of recombinant gloshedobin in cytoplasm than at 37°C.     

Soluble expression, purification, and characterization of Gloydius shedaoensis venom gloshedobin in Escherichia coli by using fusion partners

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, is usually produced as inclusion bodies in Escherichia coli cell. In this work, gloshedobin was separately fused with three fusion partners NusA, GST, and TrxA at its N terminus and then was expressed as fusion proteins in E. coli. The results showed that the NusA was the most efficient fusion partner to improve the solubility of recombinant gloshedobin. The purified NusA-fused gloshedobin with an overall yield of 64.6% was resolved as one band in the SDS-PAGE gel with molecular mass of about 90 kDa. Both fibrinogen clotting and fibrinogenolytic activities were found for the recombinant product. The purified NusA-fused gloshedobin exhibited amidolytic activity of 506 U/mg under optimal conditions of pH of 8.0 and 40°C. The inhibition study of NusA-fused gloshedobin by various inhibitors showed that serine protease inhibitors, phenylmethylsulphonyl fluoride, and N-tosyl-l-phenylalanine chloromethyl ketone, strongly inhibited its admidolytic activity, whereas ethylenediaminetetraacetic acid as well as heparin and hirudin did not, suggesting that NusA-fused gloshedobin exhibited the same characteristics as the native form of gloshedobin. The strategy of this work may contribute to improve the soluble expression level of other thrombin-like enzymes from snake venom in E. coli.     

Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis was expressed as Hsp70 fusion protein from the construct pPIC9K/hsp70-TLE in the yeast Pichia pastoris. By fusing gloshedobin to the C-terminus of Hsp70, an expression level of 44.5 mg Hsp70-gloshedobin per liter of culture was achieved by methanol induction. The fusion protein secreted in the culture medium was conveniently purified by two chromatographic steps: Q-Sepharose FF and Superdex 200. The purified enzyme had an apparent molecular mass of 98 kDa according to SDS–PAGE analysis, and exhibited fibrinogenolytic activity that preferentially degraded fibrinogen α-chain. The enzyme also degraded fibrinogen β-chain to a lesser extent, while showing no degradation toward the γ-chain. A fibrinogen clotting activity of 499.8 U/mg was achieved by the enzyme, which is within the range reported for other thrombin-like enzymes. Hsp70-gloshedobin had strong esterase activity toward the chromogenic substrate Nα-p-tosyl-Gly-Pro-Arg-p-nitroanilide, and this activity was optimal at pH 7.5 and 50 °C, and was completely inhibited by PMSF, but not by EDTA. We concluded that Hsp70 has no effect on the physiochemical and biochemical properties of gloshedobin. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. And this positive result may provide an alternative strategy for the expression of thrombin-like enzymes in microbial system.     

Structure of the HLA-A*0204 antigen,found in South American Indians. Spatial clustering of HLA-A2 subtype polymorphism

The primary structure of the HLA-A2 subtype A^*0204 (isoelectric focusing variant A2.A) has been determined. cDNA encoding this subtype was amplified by the polymerase chain reaction. Four independent full-lenght cDNA clones encoding A^*0204 were analyzed to obtain a consensus sequence for this subtype. A^*0204 differs from A^*0201 by a single nucleotide change of G to T through the coding regions, resulting in an Arg to Met change at position 97. This substitution accounts for the isoelectric focusing pattern of the subtype. The same change occurs in other HLA-A specificities in association with other changes in its vicinity. The absence of additional substitutions in A^*0204 suggests that it could have arisen from A^*0201 by point mutation, and that recurrent mutations may take place during HLA diversification. The spatial location of this change implies that A^*0204 must be a functional variant. Comparison of its sequence with other HLA-A2 subtypes reveals that much of the HLA-A2 subtype polymorphism is generated by variations in four neighboring positions, including position 97, which are located in two adjacent -strands on the floor of the peptide binding site of the molecule.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X57954. Address correspondence and offprint requests to: J. A. López de Castro.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

Luciferase cDNA from Japanese firefly,Luciola cruciata: Cloning,structure and expression in Escherichia coli

We have cloned the cDNA for luciferase from lantern poly(A)⁺ RNA of a Japanese firefly, Luciola cruciata (Genji botaru in Japanese). This cDNA directed the synthesis of enzymatically active luciferase under the control of the lac promoter in Escherichia coli. The amino acid sequence predicted from the cDNA sequence shows that Genji firefly luciferase consists of 548 amino acids and has a molecular weight of 60,024. Considerable sequence homology was found upon the comparison of the Genji and North American firefly luciferases.     

Molecular cloning of lupin leghemoglobin cDNA

Poly(A)⁺RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using³²P-labeled cDNA synthesized either from nodule poly(A)⁺RNA or from poly(A)⁺RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)⁺RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.     

CLONING OF MELITTIN cDNA FROM HONEYBEE INTO THE HIGH EXPREHION PLASMID OF ESCHERICHIA COLI*

Abstract Total mRNA from venom glands of newly emerged queen bees was reversely transcribed into cDNA and cloned into the EcoRI site of plasmid λgt11; cDNA library for bee venom was thus constructed. PCR technique was used to produce the melittin coding sequence from the cDNA library. A 87 bp product was produced and inserted into the EcoRI and PstI sites of the high level expression vector pBV220. Recombinant plasmid pBM95 was transformed into the competent cells of E.coli JM101. After screening transformants on LB medium with ampicilin, structure of the recombinant plasmid pBM95 from transformants was analyzed and melittin gene in pBM95 was sequenced. The cloned cDNA coding for honey bee melittin was obtained.     

Accidental altruism in insular pit-vipers (Gloydius shedaoensis, Viperidae)

Darwinian theory predicts that organisms will display traits that benefit themselves rather than other individuals; exceptions to this rule usually are explicable by kin selection. Our studies on an insular population of venomous snakes in north-eastern China reveal a different situation. Only one species of snake (Gloydius shedaoensis, Viperidae) occurs on the island of Shedao, and displays altruism between size (age) classes. First, small snakes frequently kill prey items larger than they can swallow themselves. This behaviour enhances rates of feeding of larger conspecifics, which scavenge the birds' carcasses. Second, large snakes kill raptorial birds (sparrowhawks Accipiter nisus) that pose little or no threat to themselves. This behaviour reduces predation risk for smaller snakes. These effects are presumably accidental consequences of the high venom toxicity of the pit-vipers, which enable them to kill inedible prey and non-threatening predators at little cost. Nonetheless, this accidental altruism may have significant ecological consequences. For example, these behaviours may contribute to the remarkably high population densities of snakes on Shedao.     

Cloning and characterization of a ribosomal protein L23a from Haemaphysalis qinghaiensis eggs by immuno screening of a cDNA expression library

A primary cDNA library with a size of 1.34 × 10⁶ PFU was constructed from Haemaphysalis qinghaiensis eggs and was immunoscreened with rabbit anti-H. qinghaiensis serum. One clone (Hq22, named following those clones obtained from adult Haemaphysalis qinghaiensis cDNA library which we constructed before) screened from the cDNA library was selected randomly for sequencing. The entire sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). A search of the cloned sequence against GenBank revealed that it related to ribosomal protein L23a (Rpl23a) and had a high percentage similarity to this protein from different species. Conserved domains for Rpl23a were also identified in the cloned sequence. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other tissues and different developmental stages of H. qinghaiensis. Based on the H. qinghaiensis Rpl23a sequence, open reading frames (ORF) of Rpl23a of Heamaphysalis longicornis and Boophilus microplus were also cloned and were performed for comparison with Rpl23a of H. qinghaiensis and other organisms as well. Vaccine based on Rpl23a recombinant protein cannot protect sheep against H. qinghaiensis.     

Molecular cloning of preprocathepsin E cDNA from the stomach of bullfrog Rana catesbeiana

A cDNA library was constructed from a poly(A)⁺ RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn¹³⁹-Leu¹⁴⁰-Thr¹⁴¹) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.     

A calmodulin-stimulated Ca-ATPase from plant vacuolar membranes with a putative regulatory domain at its N-terminus

A cDNA, BCA1, encoding a calmodulin-stimulated Ca²⁺-ATPase in the vacuolar membrane of cauliflower (Brassica oleracea) was isolated based on the sequence of tryptic peptides derived from the purified protein. The BCA1 cDNA shares sequence identity with animal plasma membrane Ca²⁺-ATPases and Arabidopsis thaliana ACA1, that encodes a putative Ca²⁺ pump in the chloroplast envelope. In contrast to the plasma membrane Ca²⁺-ATPases of animal cells, which have a calmodulin-binding domain situated in the carboxy-terminal end of the molecule, the calmodulin-binding domain of BCA1 is situated at the amino terminus of the enzyme.     

甘蔗细胞质型苹果酸脱氢酶基因的克隆及其表达分析

对甘蔗(Saccharum officinarum L.)叶片全长cDNA文库进行测序,获得了1个细胞质型苹果酸脱氢酶(cMDH)基因的全长cDNA序列,命名为Sc-cMDH。生物信息学分析表明,该基因全长1314 bp,开放阅读框为999 bp,编码332个氨基酸。Sc-cMDH与其他植物cMDH的氨基酸序列同源性高达86.5%～97.0%。Sc-cMDH包含典型的NAD+结合基元T11GAAGQI17和催化基元I184WGNH188,还有相当保守的6个半胱氨酸残基,因此推断该基因为细胞质型NAD-MDH。定量PCR分析结果表明,该基因在甘蔗叶片和根中的表达量高于茎。     

CONSTRUCTION OF A cDNA LIBRARY OF HOST RECOGNITION KAIROMONE FOR TELENOMUS THEOPHILAE*

Abstract Based on the kairomone from the accessory gland of female Bombyx mori elicting the parasitoid, Telenomus theophilae, to parasitize artifical beads. A cDNA expression library of enrich kairomone gene was constructed using mRNA‐rich secretory portion of the accessory glands of B. mori at the 9– day‐old of pupa. The titer of the unamplified library was 3.29 times 10⁴ pfu/μL with a recombinant rate of 90.05%, and titer of amplified library was 1.56 times 10⁷ pfd μL. The inserted cDNA fragments of the library ranged from 0.5 to 8.0 kb and concentrated around the 2.5 kb, 1. 1kb, and 0.75kb regions. All of the data suggest that the library has appropriate titer and high frequency kairomone gene fragments by using special developing stage and tissue of B. mori to extract mRNA. Directional cloning and λZiplox expression were help to clone the kairomone gene with immunoscreening.     

Cloning of the cDNA and genomic clones for glutathione synthetase fromArabidopsis thaliana and complementation of agsh2 mutant in fission yeast

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated fromArabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast,Schizosaccharomyces pombe, and shared only a small region of similarity with theEscherichia coli protein. A 4.3 kbSstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons.When theArabidopsis cDNA cloned in a special shuttle vector was expressed in aS. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in thegsh2 ^- mutant, and restored to substantial levels by the expression of theArabidopsis cDNA. TheS. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols,¹⁰⁹Cd²⁺ binding activity, and cadmium resistance. Since theArabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.