Functional expression of an earthworm fibrinolytic enzyme in Escherichia coli

Li, Dahui; Wei, Tong; Yang, Yongqiang

doi:10.1007/s11274-007-9515-3

Cited by 8 publications

(9 citation statements)

References 16 publications

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“…Inclusion body rCST1 did not show lytic activity which means that the refolding process is necessary for rCST1 activity. Similar findings have been reported for other lumbrokinase expressed in E.coli [26].…”

Section: Discussionsupporting

confidence: 90%

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Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

Zhang

Wang

et al. 2012

PLoS ONE

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A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida) by RT-PCR amplification. The gene designated as CST1 (GenBank No. AY840996) was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA), urokinase (uPA), and vampire bat (DSPAα1) plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3). The molecular mass of recombinant CST1 (rCST) was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7%) of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.

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Section: Discussionsupporting

confidence: 90%

“…Most of the rCST1 was found in the E. coli insoluble inclusion bodies (data now shown) which is consistent with other recombinant lumbrokinase proteins expressed in E. coli BL21(DE3)cells [19], [26]. After ProBond™ column (Invitrogen) purification, purity of the concentrated CST1 protein was evaluated by 12% SDS–PAGE analysis (Fig.…”

Section: Resultssupporting

confidence: 83%

Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

Zhang

Wang

et al. 2012

PLoS ONE

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“…Sequences for 24 LK genes have been deposited in GenBank ( Table 1 ). Only a few of these genes have been successfully expressed and characterized in E. coli [ 15 , 17 , 23 , 25 , 26 , 29 ], goat mammary glands [ 20 ], yeast Pichia pastoris [ 21 , 22 , 24 , 28 , 30 ], or plants [ 27 ]. Over the past decade, researchers have tried to produce LKs via recombinant technology; however, the majority of studies have reported that, for undetermined reasons, recombinant LKs are either not expressed or do not exhibit fibrinolytic activity.…”

Section: Engineering Lks For Potential Medical Applicationmentioning

confidence: 99%

“…The F-III-2 cDNA, with or without a native signal peptide sequence, was further studied using an E. coli expression system. Results indicated that E. coli could not recognize the native signal peptide of F-III-2 [ 25 ]. Therefore, no significant fibrinolytic activity was observed, even though the gene was expressed [ 25 ].…”

Section: Engineering Lks For Potential Medical Applicationmentioning

confidence: 99%

Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application

Wang

Tull

Cooper

et al. 2013

Evidence-Based Complementary and Alternative Medicine

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Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries for thousands of years. Oral administration of dry earthworm powder is considered as a potent and effective supplement for supporting healthy blood circulation. Lumbrokinases are a group of enzymes that were isolated and purified from different species of earthworms. These enzymes are recognized as fibrinolytic agents that can be used to treat various conditions associated with thrombosis. Many lumbrokinase (LK) genes have been cloned and characterized. Advances in genetic technology have provided the ability to produce recombinant LK and have made it feasible to purify a single lumbrokinase enzyme for potential antithrombotic application. In this review, we focus on expression systems that can be used for lumbrokinase production. In particular, the advantages of using a transgenic plant system to produce edible lumbrokinase are described.

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“…Bioactive molecules, which function as adhesions, were also found in G-90. After purification of G-90 by affinity chromatography on protein A-Sepharose (G-90/4), the structures with antigenic similarity to serum Igs were isolated [25]. Separation on SDS-PAGE resulted in four fractions (16-66 kDa) ( Fig.…”

Section: Molecules Involved In Adhesionmentioning

confidence: 99%

Earthworms as a Source of Bioactive Molecules

Grdiša¹,

Mikecin²,

Hrzenjak

2009

CBC

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Searching for bioactive molecules from natural source represents an attractive strategy. Natural bioactive molecules form the backbone of modern therapeutics. For that reason the glycolipoprotein extract (G-90) from tissue homogenate of earthworm Eisenia foetida represents a valuable source of bioactive molecules. The molecules in this preparation exhibit many biological characteristics, useful in different biological processes. This extract showed interaction with biological materials from evolutionary more developed species, such as serums, erythrocytes, normal and transformed cells in culture from different origin. The numerous biological activities in vitro and in vivo, such as mitogenicity, anticoagulation, fibrinolysis, bacteriostasis, antioxidation, were detected in G-90. In therapeutic concentrations G-90 was not toxic, allergenic, mutagen or carcinogen. G-90 protected cells in culture from oxidative damage, caused by hydrogen peroxide. It is capable to stimulate the cellular immune system. As fibrinolytic and anticoagulative agent it can participate in active maintenance of homeostasis. G-90 has the ability to stimulate the synthesis of growth factors and helps in tissue regeneration. It stimulates proliferation of fibroblasts and epithelial cells, as well as their differentiation. Accordingly, G-90 is a valuable mixture of bioactive molecules from natural source, which could be very useful in human and veterinary medicine.

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Functional expression of an earthworm fibrinolytic enzyme in Escherichia coli

Cited by 8 publications

References 16 publications

Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application

Earthworms as a Source of Bioactive Molecules

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