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.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

Homofermentative Production of d- orl-Lactate in Metabolically Engineered Escherichia coli RR1

1999 ◽  
Vol 65 (4) ◽  
pp. 1384-1389 ◽  
Author(s):  
Dong-Eun Chang ◽  
Heung-Chae Jung ◽  
Joon-Shick Rhee ◽  
Jae-Gu Pan
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Lactobacillus Casei ◽  
Lactate Production ◽  
Anaerobic Conditions ◽  
E Coli ◽  
Fermentation Product ◽  
Dehydrogenase Gene ◽  
Mutant Strains ◽  
Optically Pure

ABSTRACT We investigated metabolic engineering of fermentation pathways inEscherichia coli for production of optically pured- or l-lactate. Several pta mutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose tod-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation inppc made the mutant produce d-lactate like a homofermentative lactic acid bacterium. When the pta ppcdouble mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase ofd-lactate production, more than 62.2 g ofd-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenousl-lactate pathway, an l-lactate dehydrogenase gene from Lactobacillus casei was introduced into apta ldhA strain which lacked phosphotransacetylase andd-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to l-lactate as the major fermentation product, and up to 45 g of l-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of d-lactate, an indigenous fermentation product, or to the production of l-lactate, a nonindigenous fermentation product.

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 Purification, and activity of Human rhinovirus 3C protease fused with N-terminal GST-tag and C-terminal His-tag (GST-HRV3C-His) expressed in Escherichia coli

2019 ◽  
Vol 16 (3) ◽  
pp. 162
Author(s):  
Le Duong Vuong ◽  
Le Thi Tuong Vy ◽  
Phan Thi Phuong Trang ◽  
Nguyen Duc Hoang
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Human Rhinovirus ◽  
Purification Process ◽  
Recognition Sequence ◽  
Fusion Tag ◽  
E Coli ◽  
Specific Recognition ◽  
3C Protease ◽  
His Tag

The Human rhinovirus 3C protease (HRV3C) is one of the most effective enzymes for removing fusion tag in purification process. This protease is often produced as fusion form GST-HRV3C but there is no study about the fusion form: GST-HRV3C-His. In this study, researchers conducted the purification GST-HRV3C-His expressed in E. coli, checked the activity and investigated its application. GST-HRV3C-His could be purified using His-tag column with 86.6% purity and GST column with 96.87%. The specific activity of GST-HRV3C-His was demonstrated to be about 4500 U/mg and its application in the purification of another proteins carrying HRV3C-specific recognition sequence, LEVLFQ¯GP based on His-tag or GST-tag was also proved in this study.

Cloning and overexpression of Lactobacillus helveticus d-lactate dehydrogenase gene in Escherichia coli

1992 ◽  
Vol 208 (3) ◽  
pp. 799-805 ◽  
Author(s):  
Sunil KOCHHAR ◽  
Herbert HOTTINGER ◽  
Nathalie CHUARD ◽  
Paul G. TAYLOR ◽  
Tony ATKINSON ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Lactobacillus Helveticus ◽  
Dehydrogenase Gene
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