scholarly journals Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli

1991 ◽  
Vol 275 (1) ◽  
pp. 61-65 ◽  
Author(s):  
V D H Ding ◽  
B T Sheares ◽  
J D Bergstrom ◽  
M M Ponpipom ◽  
L B Perez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Fetal Liver ◽  
Farnesyl Diphosphate ◽  
Blue Staining ◽  
Isopentenyl Diphosphate ◽  
Native Form ◽  
Geranyl Diphosphate ◽  
Apparent Homogeneity

We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.

scholarly journals Biosynthesis of pinene in purple non-sulfur photosynthetic bacteria

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaomin Wu ◽  
Guang Ma ◽  
Chuanyang Liu ◽  
Xin-yuan Qiu ◽  
Lu Min ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Heterologous Expression ◽  
Rhodobacter Sphaeroides ◽  
Photosynthetic Bacteria ◽  
Fine Chemicals ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
Geranyl Diphosphate ◽  
Ribosome Binding ◽  
Geranyl Diphosphate Synthase

Abstract Background Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. Results Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-d-xylulose 5-phosphate synthase, 1-deoxy-d-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L. Conclusions In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

scholarly journals Molecular and Biochemical Characterization of the xlnD-Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in Pseudomonas alcaligenes NCIMB 9867

2005 ◽  
Vol 187 (22) ◽  
pp. 7696-7702 ◽  
Author(s):  
Xiaoli Gao ◽  
Chew Ling Tan ◽  
Chew Chieng Yeo ◽  
Chit Laa Poh
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Electron Donor ◽  
Biochemical Characterization ◽  
Aromatic Substrates ◽  
Gentisate Pathway ◽  
A Subunit ◽  
Pseudomonas Alcaligenes

ABSTRACT The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

scholarly journals Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

2009 ◽  
Vol 75 (22) ◽  
pp. 7291-7293 ◽  
Author(s):  
Gopal Prasad Ghimire ◽  
Hei Chan Lee ◽  
Jae Kyung Sohng
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
Streptomyces Peucetius ◽  
E Coli ◽  
Expression Of Genes ◽  
Genes Encoding

ABSTRACT Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli

2001 ◽  
Vol 358 (1) ◽  
pp. 263-268 ◽  
Author(s):  
Bernd H. A. REHM ◽  
Qingsheng QI ◽  
Br. Bernd BEERMANN ◽  
Hans-Jürgen HINZ ◽  
Alexander STEINBÜCHEL
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Inclusion Bodies ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Class Ii ◽  
Random Coil ◽  
Pha Synthase

In order to facilitate the large-scale preparation of active class II polyhydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative that contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpression of this phaC1 gene under control of the strong Ø10 promoter was achieved in Escherichia coli BL21(DE3). The fusion protein was deposited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30% of total protein. The inclusion bodies were purified by selective solubilization, resulting in approx. 70–80% pure PHA synthase, then dissolved and denatured by 6M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni2+-nitrilotriacetate–agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the chaotropic reagent. This procedure avoided the aggregation of folding intermediates which often decreases the efficiency of refolding experiments. Finally, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8m-units/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which corresponds to 27% of the maximum specific activity of the native enzyme. The refolding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the spectrum resulted in the following secondary structure prediction: 10% α-helix, 50% β-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.

scholarly journals N-acetyl-d-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1

1996 ◽  
Vol 317 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Miguel A. FERRERO ◽  
Angel REGLERO ◽  
Manuel FERNANDEZ-LOPEZ ◽  
Roberto ORDAS ◽  
Leandro B. RODRIGUEZ-APARICIO
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Amino Acid ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Neuraminic Acid ◽  
Acid Biosynthesis ◽  
Colominic Acid ◽  
Apparent Homogeneity ◽  
Escherichia Coli K1

Colominic acid is a capsular homopolymer from Escherichia coli K1 composed of α(2-8)-linked N-acetyl-d-neuraminic acid (NeuAc) residues. Recently, we have described that NeuAc synthesis in this bacterium occurs through the action of NeuAc lyase (EC 4.1.3.3) [Rodríguez-Aparicio, Ferrero and Reglero (1995) Biochem. J. 308, 501–505]. In the present work we analysed and characterized this enzyme. E. coli K1 NeuAc lyase is detected from the early logarithmic phase of growth, is induced by NeuAc and is not repressed by glucose. The enzyme was purified to apparent homogeneity (312-fold) using two types of hydrophobic chromatographies (butyl-agarose and phenyl-Sepharose CL-4B), gel filtration on Sephacryl S-200, and anion-exchange chromatography on DEAE-FPLC. The pure enzyme, whose amino acid composition and N-terminal amino acid sequence are also established, has a native molecular mass, estimated by gel filtration, of 135±3 kDa, whereas its molecular mass in SDS/PAGE was 33±1 kDa. The enzyme was able to synthesize and cleave NeuAc in a reversible reaction. The maximal rate of catalysis was achieved in 125 mM Tris/HCl buffer, pH 7.8, at 37 °C. Under these conditions, the Km values calculated for N-acetyl-d-mannosamine and pyruvate (condensation direction), and NeuAc (hydrolysis direction) were 7.7, 8.3 and 4.8 mM respectively. NeuAc synthesis by the pure enzyme was activated by Ca2+ and inhibited by Mn2+ and NeuAc, whereas the enzyme cleavage direction was inhibited by Ca2+, Mn2+ and pyruvate. The reaction products, NeuAc and pyruvate, and Ca2+ are able to regulate the direction of this enzyme (synthesis or cleavage of sialic acid) and, accordingly, to modulate colominic acid biosynthesis.

scholarly journals Biosynthesis of Pinene in Purple Non-Sulfur Photosynthetic Bacteria

2021 ◽  
Author(s):  
Xiaomin Wu ◽  
Guang Ma ◽  
Chuanyang Liu ◽  
Xin-yuan Qiu ◽  
Lu Min ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Heterologous Expression ◽  
Rhodobacter Sphaeroides ◽  
Photosynthetic Bacteria ◽  
Fine Chemicals ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
Geranyl Diphosphate ◽  
Ribosome Binding ◽  
Geranyl Diphosphate Synthase

Abstract Background: Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. Results: Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L. Conclusions: In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

scholarly journals Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1

2002 ◽  
Vol 184 (4) ◽  
pp. 1028-1040 ◽  
Author(s):  
Claudia Ehlers ◽  
Roman Grabbe ◽  
Katharina Veit ◽  
Ruth A. Schmitz
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Ammonium Assimilation ◽  
Regulatory Function ◽  
Native Form ◽  
E Coli ◽  
Methanosarcina Mazei ◽  
Apparent Homogeneity

ABSTRACT Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1 -amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.

scholarly journals Demonstration that fbiC Is Required by Mycobacterium bovis BCG for Coenzyme F420 and FO Biosynthesis

2002 ◽  
Vol 184 (9) ◽  
pp. 2420-2428 ◽  
Author(s):  
Kwang-Pil Choi ◽  
Nathan Kendrick ◽  
Lacy Daniels
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Mycobacterium Bovis ◽  
Biosynthetic Pathway ◽  
Antituberculosis Drug ◽  
Selective Agent ◽  
Coenzyme F420 ◽  
Aerobic Actinomycetes

ABSTRACT Using the nitroimidazopyran-based antituberculosis drug PA-824 as a selective agent, transposon-generated Mycobacterium bovis strain BCG (M. bovis) mutants that could not make coenzyme F420 were identified. Four independent mutants that could not make F420 or the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in the M. bovis homologue of the Mycobacterium tuberculosis gene Rv1173, which we have named fbiC. Complementation of an M. bovis FbiC− mutant with fbiC restored the F420 phenotype. These data demonstrate that fbiC is essential for F420 production and that FbiC participates in a portion of the F420 biosynthetic pathway between pyrimidinedione and FO. Homologues of fbiC were found in all 11 microorganisms that have been fully sequenced and that are known to make F420. Four of these homologues (all from members of the aerobic actinomycetes) coded for proteins homologous over the entire length of the M. bovis FbiC, but in seven microorganisms two separate genes were found to code for proteins homologous with either the N-terminal or C-terminal portions of the M. bovis FbiC. Histidine-tagged FbiC overexpressed in Escherichia coli produced a fusion protein of the molecular mass predicted from the M. bovis BCG sequence (∼95,000 Da), as well as three other histidine-tagged proteins of significantly smaller size, which are thought to be proteolysis products of the FbiC fusion protein.

scholarly journals Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli

1993 ◽  
Vol 295 (3) ◽  
pp. 719-724 ◽  
Author(s):  
A Igout ◽  
J Van Beeumen ◽  
F Frankenne ◽  
M L Scippo ◽  
B Devreese ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Hormone ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Expression System ◽  
Pituitary Hormone ◽  
Native Form ◽  
E Coli ◽  
Placental Growth Hormone

The hGH-V (or hGH-2) gene codes for human placental growth hormone (hPGH). Secretion of hPGH is continuous, in contrast with the pulsed secretion of pituitary growth hormone (hGH) which it progressively replaces in the maternal bloodstream. hGH-V cDNA has previously been cloned and isolated. Analysis of its nucleotide sequence has revealed a 191-residue protein, hPGH, differing from hGH at 13 positions. The calculated pI is more basic than that of the pituitary hormone. Here we have inserted hGH-V cDNA into the pIN-III-ompA3 plasmid in order to produce hPGH in its native form in Escherichia coli D1210. Expression of hGH-V cDNA in E. coli is significantly lower than that of hGH cDNA with the same expression system. The hPGH produced in E. coli was purified in quantities sufficient to allow its biochemical and immunochemical characterization. The molecular mass of the protein was determined by electrospray m.s. The determined mass, 22,320 Da, agrees well with the molecular mass calculated from the translated cDNA sequence, assuming the presence of two disulphide bridges. Having established the technique for producing hPGH with a primary structure identical to the natural, non-glycosylated, 22 kDa isoform, we can now plan the full physicochemical and pharmaceutical characterization of this new hormonal entity.

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