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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

Soluble expression of tPA in differentE. colistrains. After the auto-induction of tPA expression at 37°C for 24 h. The lysates from E coli, supernatant (soluble) fractions, flow-through proteins that did not bind to the column, and recombinant full-length tPA (His-tag-PSP-tPA-RGDS) were analyzed using SDS-PAGE and Coomassie blue staining. Lane 1, protein markers; lane 2, Origami 2 cell lysate; lane 3, Origami 2 supernatant; lane 4, flow-through; lane 5, elution with 200 mM imidazole from Ni2+-NTA; lane 6, BL21 lysate; lane 7, BL21 supernatant; lane 8, flow-through; lane 9, elution with 200 mM imidazole from Ni2+-NTA; lane 10, Rosetta™ 2 lysate; lane 11, Rosetta™ 2 supernatant; lane 12, flow-through; lane 13, elution with 200 mM imidazole from Ni2+-NTA.
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Fig2: Soluble expression of tPA in differentE. colistrains. After the auto-induction of tPA expression at 37°C for 24 h. The lysates from E coli, supernatant (soluble) fractions, flow-through proteins that did not bind to the column, and recombinant full-length tPA (His-tag-PSP-tPA-RGDS) were analyzed using SDS-PAGE and Coomassie blue staining. Lane 1, protein markers; lane 2, Origami 2 cell lysate; lane 3, Origami 2 supernatant; lane 4, flow-through; lane 5, elution with 200 mM imidazole from Ni2+-NTA; lane 6, BL21 lysate; lane 7, BL21 supernatant; lane 8, flow-through; lane 9, elution with 200 mM imidazole from Ni2+-NTA; lane 10, Rosetta™ 2 lysate; lane 11, Rosetta™ 2 supernatant; lane 12, flow-through; lane 13, elution with 200 mM imidazole from Ni2+-NTA.

Mentions: A reductive cytoplasmic environment and cytotoxicity are both possible obstacles for the active expression of tPA, a heterologous protein with multiple disulfide bonds, in wild-type E coli. Studier developed a reliable protocol for the lac operon/promoter-dependent auto-induction of genes in E coli [14]. The amount of human proteases expressed using auto-induction is far greater than that achieved using IPTG-based induction. In addition, supplying rare tRNAs (using the Rosetta 2 and Origami 2 strains) did not increase expression compared with BL21 (Figure 2). Origami 2 cells enhanced tPA disulfide bond formation in the cytoplasm; therefore, Origami 2 was the preferred choice for the expression of tPA.Figure 2


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)

Soluble expression of tPA in differentE. colistrains. After the auto-induction of tPA expression at 37°C for 24 h. The lysates from E coli, supernatant (soluble) fractions, flow-through proteins that did not bind to the column, and recombinant full-length tPA (His-tag-PSP-tPA-RGDS) were analyzed using SDS-PAGE and Coomassie blue staining. Lane 1, protein markers; lane 2, Origami 2 cell lysate; lane 3, Origami 2 supernatant; lane 4, flow-through; lane 5, elution with 200 mM imidazole from Ni2+-NTA; lane 6, BL21 lysate; lane 7, BL21 supernatant; lane 8, flow-through; lane 9, elution with 200 mM imidazole from Ni2+-NTA; lane 10, Rosetta™ 2 lysate; lane 11, Rosetta™ 2 supernatant; lane 12, flow-through; lane 13, elution with 200 mM imidazole from Ni2+-NTA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4379951&req=5

Fig2: Soluble expression of tPA in differentE. colistrains. After the auto-induction of tPA expression at 37°C for 24 h. The lysates from E coli, supernatant (soluble) fractions, flow-through proteins that did not bind to the column, and recombinant full-length tPA (His-tag-PSP-tPA-RGDS) were analyzed using SDS-PAGE and Coomassie blue staining. Lane 1, protein markers; lane 2, Origami 2 cell lysate; lane 3, Origami 2 supernatant; lane 4, flow-through; lane 5, elution with 200 mM imidazole from Ni2+-NTA; lane 6, BL21 lysate; lane 7, BL21 supernatant; lane 8, flow-through; lane 9, elution with 200 mM imidazole from Ni2+-NTA; lane 10, Rosetta™ 2 lysate; lane 11, Rosetta™ 2 supernatant; lane 12, flow-through; lane 13, elution with 200 mM imidazole from Ni2+-NTA.
Mentions: A reductive cytoplasmic environment and cytotoxicity are both possible obstacles for the active expression of tPA, a heterologous protein with multiple disulfide bonds, in wild-type E coli. Studier developed a reliable protocol for the lac operon/promoter-dependent auto-induction of genes in E coli [14]. The amount of human proteases expressed using auto-induction is far greater than that achieved using IPTG-based induction. In addition, supplying rare tRNAs (using the Rosetta 2 and Origami 2 strains) did not increase expression compared with BL21 (Figure 2). Origami 2 cells enhanced tPA disulfide bond formation in the cytoplasm; therefore, Origami 2 was the preferred choice for the expression of tPA.Figure 2

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