scholarly journals Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme

1995 ◽  
Vol 306 (2) ◽  
pp. 589-597 ◽  
Author(s):  
A Martinez ◽  
P M Knappskog ◽  
S Olafsdottir ◽  
A P Døskeland ◽  
H G Eiken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Host Cell ◽  
Phenylalanine Hydroxylase ◽  
Specific Activity ◽  
Factor Xa ◽  
Recombinant Enzyme ◽  
Size Exclusion ◽  
Isolation And Characterization ◽  
Human Phenylalanine Hydroxylase

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

scholarly journals Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease

1996 ◽  
Vol 313 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Anne P. DØSKELAND ◽  
Aurora MARTINEZ ◽  
Per M. KNAPPSKOG ◽  
Torgeir FLATMARK
Keyword(s):  
Fusion Protein ◽  
Phenylalanine Hydroxylase ◽  
Specific Activity ◽  
Factor Xa ◽  
Full Length ◽  
Target Sequence ◽  
Enzyme Form ◽  
Linker Peptide ◽  
Substrate Activation ◽  
Human Phenylalanine Hydroxylase

The phosphorylation of human phenylalanine hydroxylase by cyclic AMP-dependent protein kinase was studied using recombinant enzyme expressed as a fusion protein in the pMAL system of Escherichia coli. Using the target sequence of the restriction protease enterokinase (Asp4-Lys) as the linker peptide, 100% full-length human phenylalanine hydroxylase was obtained on protease cleavage. The fusion protein and human phenylalanine hydroxylase were both phosphorylated at Ser-16 with a stoichiometry of 1 mol of Pi/mol of subunit. The rate of phosphorylation of human phenylalanine hydroxylase was inhibited about 40% by the cofactor tetrahydrobiopterin, and this inhibition was completely prevented by the simultaneous presence of L-phenylalanine (i.e. at turnover conditions). Phosphorylated enzyme revealed a 1.6-fold higher specific activity than the non-phosphorylated enzyme form, and it also required a lower concentration of L-Phe for substrate activation. Preincubation with L-Phe increased the specific activity of phenylalanine hydroxylase 2- to 4-fold, L-Phe acting with positive cooperativity. Thus, the basic catalytic and regulatory properties of recombinant human phenylalanine hydroxylase, as well as those observed for the enzyme as a fusion protein, are similar to those previously reported for the rat liver enzyme. When the target sequence of the restriction protease factor Xa (Ile-Glu-Gly-Arg) was used as the linker between maltose-binding protein and human phenylalanine hydroxylase, cleavage of the fusion protein gave a mixture of full-length hydroxylase and a truncated form of the enzyme lacking the 13 N-terminal residues. Interestingly, phosphorylation of the fusion protein, before exposure to factor Xa, almost completely protected against secondary cleavage by this restriction protease at Arg-13 of phenylalanine hydroxylase.

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 Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

2010 ◽  
Vol 192 (9) ◽  
pp. 2407-2413 ◽  
Author(s):  
Jacalyn M. Green ◽  
Ryan Hollandsworth ◽  
Lenore Pitstick ◽  
Eric L. Carter
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Size Exclusion ◽  
Breakdown Product ◽  
Purification And Characterization ◽  
Sds Page

ABSTRACT The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a Km value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min−1 mg−1. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

scholarly journals Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 415 ◽  
Author(s):  
Haitao Ding ◽  
Lili Zhou ◽  
Qian Zeng ◽  
Yong Yu ◽  
Bo Chen
Keyword(s):  
Steady State ◽  
Heterologous Expression ◽  
Specific Activity ◽  
Recombinant Enzyme ◽  
Size Exclusion ◽  
Wild Type ◽  
Promising Alternative ◽  
Steady State Kinetics ◽  
Wild Type Counterpart ◽  
Exclusion Chromatography

A thermostable β-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD600 of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme β-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg−1 at 37 °C using ONPG (o-nitrophenyl-β-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3′-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with β-1,4 linkage and β-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with β-1,3 linkage.

scholarly journals Excretion of Human β-Endorphin into Culture Medium by Using Outer Membrane Protein F as a Fusion Partner in Recombinant Escherichia coli

2002 ◽  
Vol 68 (10) ◽  
pp. 4979-4985 ◽  
Author(s):  
Ki Jun Jeong ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Culture Medium ◽  
Feeding Strategy ◽  
Factor Xa ◽  
E Coli ◽  
Ph Stat ◽  
Nutrient Feeding ◽  
Outer Membrane Protein F

ABSTRACT Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage λ to construct E. coli MBEL-BL101. For the excretion of human β-endorphin as a model protein, the β-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-β-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-β-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield β-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of β-endorphin was obtained.

scholarly journals Expression of the Lactate Dehydrogenase Gene from Eptatretus okinoseanus in Escherichia coli

ISRN Zoology ◽  
2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Yoshikazu Nishiguchi ◽  
Akira Uchida ◽  
Noriko Oshima ◽  
Mitsumasa Okada
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Mass Production ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Factor Xa ◽  
Trigger Factor ◽  
Native Protein ◽  
3C Protease ◽  
Dehydrogenase Gene

Amplified Eptatretus okinoseanus cDNA was digested with NdeI and EcoRI, cloned into pCold trigger factor (TF), and transformed with Escherichia coli strain BL 21 in which a csp A promoter was introduced to inhibit the expression of foreign peptides. Recombinant lactate dehydrogenase (LDH) was obtained in the soluble fraction after sonication of the cells. The protein was digested by HRC 3C protease, thrombin, and factor Xa. The specific activity of TF-tagged protein and tagless protein were  mIU/mg and  mIU/mg, respectively. The deletion of the TF tag enhanced the activity compared with the native protein to  mIU/mg, showing that this expression method is effective for the mass production of the protein to allow further study of the structure of LDH.

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