Abstract
BackgroundHuman 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.ResultsWe 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.ConclusionsThis 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.
Highlights
Human tissue plasminogen activator belongs to the serine protease family
Rosetta host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins containing rare codons in E. coli [16]
The expression of tissue plasminogen activator (tPA) was examined in three cell lines using different Isopropyl β-D-1-Thiogalactopyranoside (IPTG) concentrations and in the absence of IPTG
Summary
Human tissue plasminogen activator (tPA) belongs to the serine protease family. The functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. The human protein comprises 527 amino acids residues, including 35 cysteine residues that participate in the functional preparation of tPA containing multiple disulfide bonds remains the bottleneck for its. Several recombinant hosts, such as Saccharomyces cerevisiae and insect systems, have been used for the industrial preparation of tPA. These have been associated with several problems including hyperglycosylation, poor export, and improper folding [7,8,9,10]
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