An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% （14 of 101） rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 （DE3） due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B （DE3）, which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.     

Expression of HSV-1 ICPO Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 （ICP0） exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICPO protein, but also the native ICPO protein with normal biological conformation.     

Expression of HSV-1 ICP0 Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.     

Establishment of a Genetic Transformation System and Its Application in Thermoanaerobacter tengcongensis

The whole-genome sequence of Thermoanaerobacter tengcongensis,an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China,was completed in 2002.However,in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system.In order to establish such a system,the plasmid pBOLOl containing the replication origin of the T.tengcongensis chromosome and a kanamycin resistance cassette,in which kanamycin resistance gene expression was controlled by the ttel482 promoter from T.tengcongensis,was constructed and introduced into T.tengcongensis via electroporation.Subsequently,the high transformation efficiency occurred when using freshly cultured T.tengcongensis cells without electroporation treatment,suggesting that T.tengcongensis is naturally competent under appropriate growth stage.A genetic transformation system for this strain was then established based on these important components,and this system was proved to be available for studying physiological characters of T.tengcongensis in vivo by means of hisG gene disruption and complementation.     

Cloning and characterization of the glutamate dehydrogenase gene inBacillus licheniformis

The gdhA genes of IRC-3 GDH strain and IRC-8 GDH strain were cloned, and they both successfully complemented the nutritional lesion of an E. coli glutamate auxotroph, Q100 GDH". However, the gdhA gene from the mutant IRC-8 GDH strain failed to complement the glutamate deficiency of the wild type strain IRC-3. The gdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression. Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I -type hexameric protein, while the GDH of Bacillus subtilis belongs to family II.     

大肠杆菌抗氟乙酸变株的选育及应用

In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E.coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon…     

Bacterial type I glutamine synthetase of the rifamycin SV producing actinomycete, Amycolatopsis mediterranei U32, is the only enzyme responsible for glutamine synthesis under physiological conditions

The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA^＋ gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.     

Expression and Immunoreactivity of a Human Group A Rotavirus Vp4

Rotavirus capsid protein Vp4 plays an important role in the virus adhering and entering the cells. In this study, a Vp4 gene cloned from a rotavirus strain TB-Chen was highly expressed in E.coli BL21 (DE3). The results of the Western blot showed that the protein possesses specific immuno-reactivities and can be specifically recognized by guinea pig antibodies against rotavirus strain SA11 or Wa. Some Vp4 dimers were formed during renaturation. These data obtained from this study provide a strong basis for further study on the structure and function of the Vp4.     

Biosynthesis of Ubiquinone in Escherichia coli K-12: Location of Genes Affecting the Metabolism of 3-Octaprenyl-4-hydroxybenzoic Acid and 2-Octaprenylphenol

Two genes (ubiB and ubiD) concerned with two successive reactions in ubiquinone biosynthesis in Escherichia coli were mapped and found to be closely linked. Mutant strains of E. coli carrying the ubiB(-) and ubiD(-) alleles were shown to accumulate 2-octaprenylphenol and 3-octaprenyl-4-hydroxybenzoic acid, respectively. These compounds were isolated and identified by using nuclear magnetic resonance and mass and infrared spectroscopy. Cell extracts from the mutant strain carrying the ubiD(-) allele lack 3-octaprenyl-4-hydroxybenzoate decarboxylase activity.     

弱氧化葡糖杆菌ddsA基因在大肠杆菌不同宿主菌中的表达

泛醌（辅酶Q）在生物体氧化呼吸链中作为重要的质子和电子传递物质。聚十异戊烯焦磷酸合成酶催化辅助酶Q10的侧链的生物合成。为了获得高产辅助酶Q10的菌株,将选择了10种不同大肠杆菌宿主菌用于弱氧化葡糖杆菌的聚十异戊烯焦磷酸合成酶基因ddsA的表达,通过产物分析证实该基因能在大肠杆菌中表达出有活性的聚十异戊烯焦磷酸合成酶,使大肠杆菌合成了辅酶Q10。此外,还发现在Escherichia coli HB101这一菌株中,ddsA的表达使辅酶Q10的产量略超过了在野生型中占主导地位的辅酶Q8的产量。该结果证明了利用大肠杆菌大规模发酵生产辅酶Q10的可能性。     

Identification of a ubiG-like gene involved in ubiquinone biosynthesis from Chlamydophila pneumoniae AR39

AIMS: To investigate if one hypothetical protein from Chlamydophila pneumoniae AR39 exerts UbiG-like function by complementary experiments. METHODS AND RESULTS: Proteins UbiG have a signature S-adenosylmethionine-binding motif compared with other methyltransferases. Probing with the conserved motif, one hypothetical protein from C. pneumoniae AR39 was proposed to be a UbiG-like protein. The protein encoding the gene was used to swap its counterpart in Escherichia coli, and its expression in resultant strain DYCG was confirmed by RT-PCR. Strain DYCG grew on succinate as a carbon source, and rescued ubiquinone content in vivo, while the ubiG deletion strain DYK did not. CONCLUSIONS: Results indicate that the putative protein from C. pneumoniae exerts a UbiG-like function involved in ubiquinone biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of the ubiG-like gene will facilitate research on ubiquinone biosynthesis and aerobic respiration in the genus Chlamydophila owing to the important function of ubiquinone in vivo.     

Ubiquinone biosynthesis in Saccharomyces cerevisiae. Isolation and sequence of COQ3, the 3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase gene

Ubiquinone (or coenzyme Q) is a lipid component of the respiratory chain in the inner mitochondrial membrane, in which it functions in electron transport. Recent reports show that ubiquinone and ubiquinone biosynthetic enzymes are present in both mitochondrial and nonmitochondrial membranes of cells (Kalen, A., Appelkvist, E.-L., Chojnacki, T., and Dallner, G. (1990) J. Biol. Chem. 265, 1158-1164) although the functions that ubiquinone may play outside of the mitochondrion are not understood. To study coenzyme Q synthesis and function we cloned the 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase gene by functional complementation of a yeast coenzyme Q mutant strain, defective in the COQ3 gene (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225). This gene restores both coenzyme Q synthesis in the mutant strain and the ability to grow on media containing glycerol, a nonfermentable substrate. A one-step in situ gene replacement with the cloned DHHB methyltransferase DNA directs integration to the yeast COQ3 locus on chromosome XV of Saccharomyces cerevisiae, establishing that the COQ3 locus encodes the DHHB methyltransferase structural gene. The predicted amino acid sequence of the yeast DHHB methyltransferase contains a methyltransferase consensus sequence and shows a 40% identity with an open reading frame of Escherichia coli, the gyrA5' hypothetical protein. This open reading frame is adjacent to the gyrA gene and close to the mapped location of the ubiG gene at 48 min on the E. coli chromosome. These results suggest that the E. coli gyrA5' open reading frame encodes a methyltransferase and may correspond to the ubiG gene, which is required for ubiquinone biosynthesis.     

Regulation of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli

The expressions of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli were compared under a variety of growth conditions and in several genetic backgrounds. LacZ operon fusions were constructed and were inserted in single copies into strain MC4100 and into its fnr, arcA or hemA carrying derivatives. During aerobic growth the expressions of both ubiD and ubiX depended on the carbon source: succinate>glycerol>glucose. Mutations in fnr, arcA or hemA increased the expressions of both genes. During anaerobic growth in LB medium glucose strongly inhibited the expression of ubiD but not of ubiX.     

Identification of the ubiD gene on the Escherichia coli chromosome

The open reading frame at 86.7 min on the Escherichia coli chromosome, "yigC," complemented a ubiD mutant strain, AN66, indicating that yigC is the ubiD gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.     

Heteromer formation of a long-chain prenyl diphosphate synthase from fission yeast Dps1 and budding yeast Coq1

Ubiquinone is an essential factor for the electron transfer system and is also a known lipid antioxidant. The length of the ubiquinone isoprenoid side-chain differs amongst living organisms, with six isoprene units in the budding yeast Saccharomyces cerevisiae, eight units in Escherichia coli and 10 units in the fission yeast Schizosaccharomyces pombe and in humans. The length of the ubiquinone isoprenoid is determined by the product generated by polyprenyl diphosphate synthases (poly-PDSs), which are classified into homodimer (i.e. octa-PDS IspB in E. coli) and heterotetramer [i.e. deca-PDSs Dps1 and D-less polyprenyl diphosphate synthase (Dlp1) in Sc. pombe and in humans] types. In this study, we characterized the hexa-PDS (Coq1) of S. cerevisiae to identify whether this enzyme was a homodimer (as in bacteria) or a heteromer (as in fission yeast). When COQ1 was expressed in an E. coli ispB disruptant, only hexa-PDS activity and ubiquinone-6 were detected, indicating that the expression of Coq1 alone results in bacterial enzyme-like functionality. However, when expressed in fission yeast Deltadps1 and Deltadlp1 strains, COQ1 restored growth on minimal medium in the Deltadlp1 but not Deltadps1 strain. Intriguingly, ubiquinone-9 and ubiquinone-10, but not ubiquinone-6, were identified and deca-PDS activity was detected in the COQ1-expressing Deltadlp1 strain. No enzymatic activity or ubiquinone was detected in the COQ1-expressing Deltadps1 strain. These results indicate that Coq1 partners with Dps1, but not with Dlp1, to be functional in fission yeast. Binding of Coq1 and Dps1 was demonstrated by coimmunoprecipitation, and the formation of a tetramer consisting of Coq1 and Dps1 was detected in Sc. pombe. Thus, Coq1 is functional when expressed alone in E. coli and in budding yeast, but is only functional as a partner with Dps1 in fission yeast. This unusual observation indicates that different folding processes or protein modifications in budding yeast/E. coli versus those in fission yeast might affect the formation of an active enzyme. These results provide important insights into the process of how PDSs have evolved from homo- to hetero-types.     

Sulfide oxidation in gram-negative bacteria by expression of the sulfide-quinone reductase gene of Rhodobacter capsulatus and by electron transport to ubiquinone.

The oxidation of sulfide was studied in recombinant bacteria expressing the sulfide-quinone reductase gene (sqr) from Rhodobacter capsulatus. Sulfide was oxidized by the Escherichia coli strain W3110 harboring the sqr construct (pKKSQ) under anaerobic conditions and nitrate was utilized as a terminal electron acceptor. Following the oxidation, elemental sulfur and nitrite were produced as the final reaction products. This activity was retained in the membrane preparation and was sensitive towards antimycin A, stigmatellin, and azide. As a consequence of the ubiquinone deficiency, this activity was markedly decreased. In additon, by recovery of ubiquinone, the oxidation was also restored to rates similar to those of the wild-type strain. These results indicate that sulfide oxidation in this strain occurs via the quinone pool in vivo, and that this sulfide-quinone reductase (SQR) in particular utilizes ubiquinone as a more appropriate electron acceptor than menaquinone or demetylmenaquinone. To our knowledge, this is the first study to show a direct interaction between SQR and ubiquinone in cells. When expressed in Pseudomonas putida and Rhizobium meliloti, the SQR conferred on these organisms the ability to oxidize sulfide as well as E. coli in vivo.     

Alternative hydroxylases for the aerobic and anaerobic biosynthesis of ubiquinone in Escherichia coli.

The synthesis of ubiquinone under anaerobic conditions was examined in a variety of strains of Escherichia coli K12. All were shown to synthesize appreciable quantities of ubiquinone 8 when grown anaerobically on glycerol in the presence of fumarate. Under these conditions, ubiquinone 8 was in most cases the principal quinone formed, and levels in the range 50--70% of those obtained aerobically were observed. Studies with mutants blocked in the various reactions of the aerobic pathway for ubiquinone 8 synthesis established that under anaerobic conditions three alternative hydroxylation reactions not involving molecular oxygen are used to derive the C-4, -5, and -6 oxygens of ubiquinone 8. Thus, mutants blocked in either of the three hydroxylation reactions of the aerobic pathway (ubiB, ubiH, or ubiF) are each able to synthesize ubiquinone 8 anaerobically, whereas mutants lacking the octaprenyltransferase (ubiA), carboxy-lyase (ubiD), or methyltransferases (ubiE or ubiG) of the aerobic pathway remain blocked anaerobically. The demonstration that E. coli possesses a special mechanism for the anaerobic biosynthesis of ubiquinone suggests that this quinone may play an important role in anaerobic metabolism.