Engineering E. coli to Produce Human Urokinase
Every year, millions of lives are threatened by thrombotic diseases—heart attacks, strokes, and pulmonary embolisms—where blood clots obstruct vital blood vessels. The body's natural defense against these clots is a process called fibrinolysis, an intricate biological system responsible for breaking down fibrin, the main structural component of blood clots.
At the heart of this system are remarkable enzymes called plasminogen activators, which initiate the dissolution of clots before they can cause irreparable damage.
For decades, scientists have sought to harness these natural clot-busting enzymes for medicine. One of the most promising of these is urokinase, a human protein that efficiently activates plasminogen to form plasmin, the enzyme that directly degrades fibrin clots.
Urokinase was first discovered in human urine, which is how it got its name.
Amino acids in urokinase
Molecular weight
Disulfide bonds
Urokinase is a sophisticated serine protease with a complex three-dimensional structure that's essential to its function. The molecule is composed of 411 amino acids arranged in three distinct domains that must fold precisely into the correct configuration 9 .
Perhaps most challenging are the multiple disulfide bonds—specific cysteine residues that must connect in exactly the right pattern to stabilize the protein's structure.
E. coli has long been the workhorse of molecular biology, but its cellular environment is fundamentally different from human cells:
Early attempts to express urokinase in E. coli resulted in misfolded, biologically inactive protein trapped in inclusion bodies 7 , requiring complex extraction and refolding procedures with poor yields.
The human urokinase gene was optimized for bacterial expression with codon preferences that maximize production in E. coli. This optimized gene was inserted into specialized expression vectors containing strong promoters and selection markers 7 .
Researchers experimented with different cellular locations:
A critical breakthrough came from co-expressing urokinase with DsbC, a bacterial enzyme that catalyzes disulfide bond isomerization, dramatically increasing production of active protein 3 .
Multiple chromatography methods were employed:
Low yield of active protein
High yield of active protein
| Enzyme | Km (μM) | kcat (s⁻¹) | Catalytic Efficiency (kcat/Km, μM⁻¹s⁻¹) | Source |
|---|---|---|---|---|
| Pro-urokinase | Not specified | Not specified | 0.05 | 1 |
| Urokinase | Not specified | Not specified | 0.02 | 1 |
| tPA (with DsbC) | Comparable to native tPA | Comparable to native tPA | Nearly identical to authentic tPA | 3 |
Essential reagents for recombinant urokinase research
| Reagent/Category | Specific Examples | Function in Research |
|---|---|---|
| Expression Vectors | pET22b, pBAD vectors | Provide genetic backbone for urokinase gene with strong promoters and selection markers |
| Signal Peptides | PelB sequence | Directs expressed urokinase to the periplasmic space for better folding |
| Specialized E. coli Strains | Origami B(DE3), SF110 | Engineered strains with enhanced disulfide bond formation capabilities |
| Disulfide Bond Enzymes | DsbA, DsbC | Catalyze formation and isomerization of disulfide bonds in expressed proteins |
| Refolding Reagents | Urea, Guanidine HCl, Glutathione | Solubilize and refold urokinase from inclusion bodies into active form |
| Chromatography Media | Lysine-Sepharose, Hydroxylapatite | Purify urokinase from other bacterial proteins based on specific binding properties |
| Activity Assay Components | Plasminogen, Gelatin, Chromogenic substrates | Measure enzymatic activity and specificity of recombinant urokinase |
| Component | Concentration | Function |
|---|---|---|
| Urea | 2-4 M | Mild denaturant for solubility |
| Reduced Glutathione | 1-5 mM | Reducing environment |
| Oxidized Glutathione | 0.1-1 mM | Oxidative component |
| L-Arginine | 0.5-1 M | Suppresses aggregation |
| CHAPS | 0.1-0.5% | Improves solubility |
Co-expression of DsbC increased yield of active tPA to ~180 μg/L in high-cell-density fermentation 3
The successful expression of biologically active urokinase in E. coli represents a triumph of molecular biology and biotechnology. What began as a challenging scientific problem has evolved into sophisticated methodology that has paved the way for producing many therapeutically important proteins.
The strategies developed—including periplasmic targeting, disulfide bond engineering, and sophisticated refolding protocols—have created a toolbox that scientists now use to produce countless complex proteins. These advances have contributed to the "fibrinolysis renaissance" noted by researchers 6 , renewing interest in fibrinolytic therapy and leading to new generations of thrombolytic agents.
Engineered variants with improved properties such as greater fibrin specificity and resistance to natural inhibitors .
Cutting-edge approaches include clot-targeted nanoparticles and other sophisticated systems 4 .
Recombinant fibrinolytic enzymes are being explored for applications beyond traditional thrombosis treatment 4 .
The story of urokinase production in E. coli exemplifies how creative problem-solving in basic science can lead to technologies with profound impacts on human health. As research continues, these advances promise to deliver ever more effective therapies for the millions of patients affected by thrombotic diseases worldwide.