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Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli.

Long X, Gou Y, Luo M, Zhang S, Zhang H, Bai L, Wu S, He Q, Chen K, Huang A, Zhou J, Wang D - BMC Biotechnol. (2015)

Bottom Line: The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium).The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.This is the first report that describes the heterologous expression of correctly folded active full-length tPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China. 582526247@qq.com.

ABSTRACT

Background: Human tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. Human tPA is composed of 527 amino acids residues and contains 17 disulfide bonds. Escherichia coli has been used only rarely for the efficient production of recombinant tPA. However, the functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. Here, we describe the soluble expression and characterization of full-length tPA by auto-induction in E. coli.

Results: We achieved optimal levels of gene expression, minimized negative effects related to the production of heterologous proteins, and optimized cytoplasmic yields. Three different E. coli strains, BL21 (DE3), Rosetta, and Origami 2, could express tPA using an auto-induction mechanism. In addition, similar yields of recombinant protein were produced at temperatures of 33, 35, and 37°C. The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium). The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.

Conclusions: This is the first report that describes the heterologous expression of correctly folded active full-length tPA. This could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels without in vitro refolding during the production step.

No MeSH data available.


Related in: MedlinePlus

Lysis on a fibrin plate using urokinase and tPA. tPA-RGDS; HisTag-PSP-tPA-RGDS; urokinase standard (10 × 103 IU ml−1. positive control); BSA (bovine serum albumin standard, negative control). The fibrinolytic activity of tPA was measured and compared with urokinase (positive control) and bovine serum albumin (BSA; negative control). A, dot 1, 10 μg BSA; dot 2, 2.5 × 103 IU urokinase; dot 3, 5.0 × 103 IU urokinase (positive control); dot 4, 10 μg His-tag-PSP-tPA-RGDS; dot 5, 20 μg His-tag-PSP-tPA-RGDS; dot 6, 10 μg tPA-RGDS; dot 7, 20 ug tPA-RGDS. B, the diameters of the lysis dots on the fibrin plate from at four independent evaluations. The bars indicate standard error of the mean.
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Fig6: Lysis on a fibrin plate using urokinase and tPA. tPA-RGDS; HisTag-PSP-tPA-RGDS; urokinase standard (10 × 103 IU ml−1. positive control); BSA (bovine serum albumin standard, negative control). The fibrinolytic activity of tPA was measured and compared with urokinase (positive control) and bovine serum albumin (BSA; negative control). A, dot 1, 10 μg BSA; dot 2, 2.5 × 103 IU urokinase; dot 3, 5.0 × 103 IU urokinase (positive control); dot 4, 10 μg His-tag-PSP-tPA-RGDS; dot 5, 20 μg His-tag-PSP-tPA-RGDS; dot 6, 10 μg tPA-RGDS; dot 7, 20 ug tPA-RGDS. B, the diameters of the lysis dots on the fibrin plate from at four independent evaluations. The bars indicate standard error of the mean.

Mentions: Disulfide bonds, which play multiple critical roles in protein stability and function, are often abundantly present in secreted proteins. The functional expression of human proteins with multiple disulfide bonds, such as tPA, in bacterial systems has proved challenging. Therefore, we used fibrin plate assays to assess whether the recombinant tPA was actively folded. Plasminogen was used as the tPA substrate in the medium on the plate. Active tPA can bind to plasminogen and cleave it into plasmin, which degrades fibrin and results a clear lysed zone on the fibrin/agar plate. RGDS, as the binding motif of integrin, will improve the specific affinity of the recombinant tPA to substrate [15]. Similar to tPA, Urokinase could cleave plasminogen into plasmin and was used as a positive control [19]. As is shown in Figure 6A and B, tPA and urokinase exhibited similar halo patterns, suggesting that the His-tag-PSP and RGDS, which were attached to the C- and N-terminus of tPA, respectively, did not affect either tPA receptor-ligand binding or the resulting signal transduction. In addition, the similar diameter of the halo patterns of HisTag-PSP-tPA-RGDS and tPA-RGDS suggests that they exhibit similar amidolytic activities in vitro. The activity of recombinant tPA was similar to that of urokinase.Figure 6


Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli.

Long X, Gou Y, Luo M, Zhang S, Zhang H, Bai L, Wu S, He Q, Chen K, Huang A, Zhou J, Wang D - BMC Biotechnol. (2015)

Lysis on a fibrin plate using urokinase and tPA. tPA-RGDS; HisTag-PSP-tPA-RGDS; urokinase standard (10 × 103 IU ml−1. positive control); BSA (bovine serum albumin standard, negative control). The fibrinolytic activity of tPA was measured and compared with urokinase (positive control) and bovine serum albumin (BSA; negative control). A, dot 1, 10 μg BSA; dot 2, 2.5 × 103 IU urokinase; dot 3, 5.0 × 103 IU urokinase (positive control); dot 4, 10 μg His-tag-PSP-tPA-RGDS; dot 5, 20 μg His-tag-PSP-tPA-RGDS; dot 6, 10 μg tPA-RGDS; dot 7, 20 ug tPA-RGDS. B, the diameters of the lysis dots on the fibrin plate from at four independent evaluations. The bars indicate standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4379951&req=5

Fig6: Lysis on a fibrin plate using urokinase and tPA. tPA-RGDS; HisTag-PSP-tPA-RGDS; urokinase standard (10 × 103 IU ml−1. positive control); BSA (bovine serum albumin standard, negative control). The fibrinolytic activity of tPA was measured and compared with urokinase (positive control) and bovine serum albumin (BSA; negative control). A, dot 1, 10 μg BSA; dot 2, 2.5 × 103 IU urokinase; dot 3, 5.0 × 103 IU urokinase (positive control); dot 4, 10 μg His-tag-PSP-tPA-RGDS; dot 5, 20 μg His-tag-PSP-tPA-RGDS; dot 6, 10 μg tPA-RGDS; dot 7, 20 ug tPA-RGDS. B, the diameters of the lysis dots on the fibrin plate from at four independent evaluations. The bars indicate standard error of the mean.
Mentions: Disulfide bonds, which play multiple critical roles in protein stability and function, are often abundantly present in secreted proteins. The functional expression of human proteins with multiple disulfide bonds, such as tPA, in bacterial systems has proved challenging. Therefore, we used fibrin plate assays to assess whether the recombinant tPA was actively folded. Plasminogen was used as the tPA substrate in the medium on the plate. Active tPA can bind to plasminogen and cleave it into plasmin, which degrades fibrin and results a clear lysed zone on the fibrin/agar plate. RGDS, as the binding motif of integrin, will improve the specific affinity of the recombinant tPA to substrate [15]. Similar to tPA, Urokinase could cleave plasminogen into plasmin and was used as a positive control [19]. As is shown in Figure 6A and B, tPA and urokinase exhibited similar halo patterns, suggesting that the His-tag-PSP and RGDS, which were attached to the C- and N-terminus of tPA, respectively, did not affect either tPA receptor-ligand binding or the resulting signal transduction. In addition, the similar diameter of the halo patterns of HisTag-PSP-tPA-RGDS and tPA-RGDS suggests that they exhibit similar amidolytic activities in vitro. The activity of recombinant tPA was similar to that of urokinase.Figure 6

Bottom Line: The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium).The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.This is the first report that describes the heterologous expression of correctly folded active full-length tPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China. 582526247@qq.com.

ABSTRACT

Background: Human tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. Human tPA is composed of 527 amino acids residues and contains 17 disulfide bonds. Escherichia coli has been used only rarely for the efficient production of recombinant tPA. However, the functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. Here, we describe the soluble expression and characterization of full-length tPA by auto-induction in E. coli.

Results: We achieved optimal levels of gene expression, minimized negative effects related to the production of heterologous proteins, and optimized cytoplasmic yields. Three different E. coli strains, BL21 (DE3), Rosetta, and Origami 2, could express tPA using an auto-induction mechanism. In addition, similar yields of recombinant protein were produced at temperatures of 33, 35, and 37°C. The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium). The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.

Conclusions: This is the first report that describes the heterologous expression of correctly folded active full-length tPA. This could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels without in vitro refolding during the production step.

No MeSH data available.


Related in: MedlinePlus