Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain

Mohammadi, Nasrin; Bandehpour, Mojgan; Sotoodehnejadnematalahi, Fattah; Kazemi, Bahram

doi:10.1002/prot.26275

Cited by 3 publications

(3 citation statements)

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“…According to the Jonebring et al study, the highest yield of CHO- produced ecarin was up to 7000 EU ecarin/litre in lab scale shaker cultures (equivalent to ~5500 mU/L), but the recovery percentage of the purified rEcarin was not reported [ 13 ]. Very recently, Mohammadi et al reported that the ecarin metalloproteinase domain was expressed in E. coli , with high plasma clotting activity, but it is unclear whether expression of the metalloproteinase domain could be scaled up to produce high yield [ 17 ]. In our study, we obtained a purification recovery of 76% of rEcrain and the yield of the purified rEcarin was 18.7 mg/L, with the prothrombin activity at 6100 mU/L (7820 IU/L).…”

Section: Discussionmentioning

confidence: 99%

“…However, the use of ecarin purified from snake venoms has many of the drawbacks of OsPA and PtPA; therefore, it is desirable to have a recombinantly expressed form. The cloning and expression of ecarin in active form, including within the metalloproteinase domain, have been previously described in different systems [ 13 , 17 , 18 , 19 ]. However, it has not been demonstrated whether these recombinant forms can be scaled up to produce a commercial supply of recombinant ecarin.…”

Section: Introductionmentioning

confidence: 99%

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Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics

et al. 2022

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We describe here the purification and cloning of a codon-optimized form of the snake prothrombin activator ecarin from the saw scaled viper (Echis carinatus) expressed in mammalian cells. Expression of recombinant ecarin (rEcarin) was carried out in human embryonic kidney cells (HEK) cells under conditions for the development and performance of a novel and scalable recombinant snake ecarin to industry standards. Clotting performance of the rEcarin was established in recalcified citrated whole blood, plasma, and fresh whole blood and found to be comparable to native ecarin (N-Ecarin). Furthermore, hemolysis was observed with N-Ecarin at relatively high doses in both recalcified citrated and fresh whole blood, while clotting was not observed with rEcarin, providing an important advantage for the recombinant form. In addition, rEcarin effectively clotted both recalcified citrated whole blood and fresh whole blood containing different anticoagulants including heparin, warfarin, dabigatran, Fondaparinux, rivaroxaban and apixaban, forming firm clots in the blood collection tubes. These results demonstrate that rEcarin efficiently clots normal blood as well as blood spiked with high concentrations of anticoagulants and has great potential as an additive to blood collection tubes to produce high quality serum for analyte analysis in diagnostic medicine.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics

et al. 2022

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“…The aim of this study was to produce safe, cost-effective, and high-performance r-Ecarin for diagnostic, and therapeutic purposes. Comparing the expression and function of full-length and truncated forms of this protein obtained from the previous study (19) helps to select the right one for production.…”

Section: Objectivesmentioning

confidence: 99%

Cloning, Expression and Purification of Full-length Recombinant Ecarin and Comparing Its Expression and Function with Its Truncated Form

et al. 2022

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: Ecarin is a metalloproteinase found in snake venom (SVMP) with an important role in coagulation and control of hemostasis. It can specifically produce active-thrombin from prethrombin-2 and does not differentiate between normal and abnormal prothrombin. It is used in diagnostic tests and to evaluate the treatment process of many diseases. There are many drawbacks associated with separating these compounds from snake venom. Therefore, in this study, full-length recombinant Ecarin (r-Ecarin) was cloned, expressed, and purified in eukaryotic host cells. To determine the most effective form of the enzyme, r-Ecarin was compared with the recombinant truncated form, which has only the metalloprotease domain of the protein (r-Ecamet) in terms of function and expression. Briefly, A DNA construct composed of sequence-encoding Ecarin was designed and cloned into pCAGGS expression vector and, subsequently, expressed in Chinese Hamster Ovary (CHO) cells. To identify the enzymatic activity of expressed protein, a bioactivity assay was performed. Blood coagulation time and expression levels of r-Ecarin and r-Ecamet proteins were compared. Also, a histopathological assessment was carried out on the liver of mice treated with these proteins. Comparison of r-Ecarin and r-Ecamet expression pattern demonstrated that full-length Ecarin expression has at least 2-fold higher expression in eukaryotic cells. Determination of r-Ecarin function proved that this protein is capable of prothrombin cleavage and producing thrombin. Comparison of PT test results between the r-Ecarin and r-Ecamet showed that there is a significant difference in the activity of the two enzymes and the full-length protein coagulates the blood in less time.

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Hepatocyte growth factor (HGF) gene: molecular characterisation of complete coding sequence and expression profile in Tarim red deer (Cervus hanglu yarkandensis) antlers

Lin,

Wang,

Rui

et al. 2024

Anim. Prod. Sci.

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Context The cDNA sequence of hepatocyte growth factor (HGF) gene in Tarim red deer has not been reported yet. Aims This study aims to obtain the full-length cDNA sequence of HGF and analyse its expression in different parts of developing antler tissues. The result provides foundational data for understanding the potential role of HGF in regulating antler growth and development. Methods Rapid amplification of cDNA ends was used to obtain the full-length cDNA sequence of Tarim red deer HGF. The pET28a (+) vector was constructed for prokaryotic expression of the recombinant protein in E. coli BL21 (DE3). The expression of HGF in different antler tissues was detected using real-time quantitative polymerase chain reaction and immunohistochemistry. Key results The full-length cDNA of Tarim red deer HGF was found to consist of a 156 bp 5’untranslated region (UTR), a 112 bp 3’UTR, and a 2193 bp open reading frame encoding a protein of 730 amino acids. The recombinant HGF protein expressed in prokaryotic cells formed inclusion bodies. HGF and its receptor c-Met were expressed in all four different antler tissues, with the highest expression level in velvet skin, followed by bone, cartilage, and the lowest in the mesenchyme. Conclusions The study successfully obtained the full-length cDNA sequence of Tarim red deer HGF and identified the expression profile of HGF and c-Met in different antler tissues. HGF is a candidate gene that may play a role in regulating the growth and development of deer antler. Implications These findings provide fundamental data for further investigations into the role of HGF in antler development. Understanding the function of HGF in antler development could have implications for elucidating the mechanism of antler regeneration.

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Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain

Cited by 3 publications

References 22 publications

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics

Cloning, Expression and Purification of Full-length Recombinant Ecarin and Comparing Its Expression and Function with Its Truncated Form

Hepatocyte growth factor (HGF) gene: molecular characterisation of complete coding sequence and expression profile in Tarim red deer (Cervus hanglu yarkandensis) antlers

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