DsbC is evident as an intense Coomassie blue-stained band. D Western blot of tPA expression in different strains. In contrast, the coexpression of high levels of DsbC in cells grown in either rich or minimal medium resulted in a dramatic increase in plasminogen activation Fig.
Zymography revealed that this activity arose from a band with a electrophoretic mobility slightly less than that of the single-chain tPA standard Fig. This is consistent with the fact that the tPA standard is glycosylated whereas the bacterially expressed protein is not. No other bands were detectable in zymography gels Fig. However, because the trc promoter is leaky, DsbC synthesis occurred even in uninduced cultures, resulting in low but detectable tPA activities. In fact, the addition of as little as 0.
DsbA also stimulated the formation of active tPA, but the specific activity obtained was fold lower than that of cultures expressing DsbC. The addition of 2 mM GSH, 2. DsbC exhibits disulfide isomerase activity in vitro, and there is strong evidence that it has a similar role in vivo 19 , 24 , DsbC can function as an isomerase only when its active-site cysteine thiols are reduced, presumably by the membrane protein DsbD DipZ 25 , In vitro, DsbC is oxidized readily by DsbA, the primary catalyst of cysteine oxidation in the periplasmic space Bardwell and coworkers showed that substitution of the residues within the Cys-X-X-Cys active-site motif of DsbA modulates the redox potential of the protein 9.
Several DsbA active-site mutants with a range of redox potentials were coexpressed together with tPA in a dsbA null mutant strain background. However, no improvement in tPA activity was observed relative to cells transformed with a control plasmid containing the wild-type dsbA gene. Similarly, coexpression of DsbC in a set of strains containing chromosomally integrated dsbA mutants did not result in any further increase in active tPA 3a. The growth rate was maintained at 0.
Preliminary experiments revealed that vigorous induction with 2 mM IPTG of DsbC expression, followed by induction of tPA expression, led to a dramatic reduction in the oxygen uptake rate after about 1.
Growth ceased, and a slow decline in the OD of the culture soon followed. Therefore, a lower concentration of IPTG 0. IPTG was added when the culture reached an OD of around 80; this was followed by a bolus of arabinose 30 min later.
Under these conditions, the oxygen uptake rate remained constant for 3. The maximum specific tPA activity was attained 2. The peak specific activity obtained in the fermentor was essentially identical to that in shaker flasks.
Approximately 25 g of cell protein was obtained per liter of culture. Production of tPA in a high-cell-density fermentation. The cell density, reported as OD , and the oxygen uptake rate OUR are shown together with the tPA specific activity data from samples taken 0. The tPA activity was determined by the indirect chromogenic assay. Clarified cell lysates were loaded onto an l -lysine—Sepharose column. The bound tPA was eluted with 0. The eluant from the l -lysine—Sepharose affinity chromatography step was loaded onto a second column containing immobilized E.
Active tPA was eluted with 0. The bacterial tPA became bound irreversibly to ultrafiltration membranes and therefore could not be concentrated in this manner from the ETI column eluant.
For this purpose and also to remove two contaminating E. Interestingly, although the bacterial tPA bound quantitatively to ETI, it could be eluted from the resin with a lower concentration of KSCN relative to the glycosylated, two-chain protein 0.
Consequently, it appears that glycosylation affects the equilibrium dissociation constant for ETI. We have identified conditions that allow the production of significant amounts of active, full-length tPA in E. Normally, the formation of disulfide bonds takes place in the periplasmic space. In this study tPA was fused to the stII leader peptide, which was efficient in directing the mature protein into the periplasmic space.
The stII leader peptide does not appear to be unique, and a similar yield of proteolytically active tPA was obtained when the tPA gene was fused to the OmpA leader peptide 29a.
As discussed in Results and as shown in Fig. To the best of our knowledge, this dramatic increase in the production of active tPA represents by far the most striking improvement in the folding of a foreign protein ever obtained via the coexpression of foldases 8. The rate-limiting step in the oxidative folding of eukaryotic proteins in the periplasmic space appears to be the isomerization of mismatched disulfides Reduced DsbC has been shown to be an efficient catalyst of disulfide bond isomerization in vitro 6.
Moreover, recent studies have shown that DsbC is maintained primarily in a reduced state in vivo, suggesting that its primary role in the cell is the catalysis of disulfide isomerization 17 , 31 , The dramatic increase in the folding of tPA in cells coexpressing DsbC is consistent with this hypothesis.
We believe that the high level of DsbC increased the disulfide isomerization capacity of the periplasmic space, thus facilitating the rearrangement of incorrect disulfides in nascent tPA. Indeed, Joly et al. In this case, overexpression of DsbC increased the total yield of IGF-1 but not the amount of soluble active protein. However, this result may be a consequence of the very high level of IGF-1 overexpression 7.
An alternative explanation for our results is that DsbC did not participate directly in the folding of tPA. Instead, its overexpression simply resulted in a higher concentration of protein thiols, thereby altering the redox potential of the periplasm to favor the formation of correct disulfide bonds. However, this hypothesis can be ruled out since neither the overexpression of other cysteine oxidoreductases nor the manipulation of the redox potential of the periplasm through the addition of GSH or GSSG had an effect similar to that of DsbC.
A low level of active tPA was detected in cells coexpressing DsbA, but this effect could not be further enhanced by the addition of reduced or oxidized glutathione. Chromosomally expressed DsbA is found almost exclusively in the oxidized form and is a potent catalyst of disulfide bond formation It is possible that when DsbA is overexpressed, a fraction of the protein fails to be oxidized by DsbB and, instead, is present in the reduced form that can catalyze disulfide bond isomerization 16 , This activity of the reduced DsbA may in turn be responsible for the small levels of active tPA.
Overexpression of rPDI has been shown to increase the yield of small heterologous proteins 15 , 28 , but it had a very minor effect on tPA. This may be because rPDI functions as a protein thiol oxidase in the bacterial periplasmic space but was shown to be rather ineffective in catalyzing the rate-limiting isomerization in BPTI in the periplasmic space of E.
The highest level of active tPA was observed when the synthesis of DsbC from a trc promoter was induced first, followed by the induction of tPA expression 30 min later. Interestingly, high-level induction of DsbC, but not DsbA, rPDI, or tPA alone, was found to be particularly toxic, resulting in cessation of growth within 3 to 4 h after induction. The precursor form of DsbC was found to accumulate in the cell, raising the possibility that the observed toxicity is linked to the saturation of the protein translocation machinery.
To obtain the maximum tPA specific activity in a high-cell-density fermentation, it was necessary to use a low concentration of IPTG, which allowed the production phase to be prolonged. Also, maintaining a low growth rate through the slow feeding of glucose was found to be essential in order to attain a high cell density in the fermentor. SF was found to be a prolific producer of acetate, which accumulated to inhibitory levels when glucose feeding was adjusted to control the growth rate above 0.
The above observations suggest a number of ways in which the expression of active, full-length tPA can be increased further. First, in the present study the level of accumulation of tPA was low regardless of the promoter used. A higher level of tPA synthesis may be beneficial and could be obtained by optimizing translation initiation and by substitution of rare codons in the tPA gene 4.
Second, since DsbC overexpression has been found to inhibit growth, conditions that inhibit DsbC toxicity will have to be identified to prolong the production phase and help achieve even higher cell densities.
The ability to produce substantial amounts of a heterologous protein as complex as tPA in E. When protein glycosylation is not essential, expression in bacteria is clearly advantageous in terms of both cost and simplicity. We are grateful to S. About one third of these subjects exhibited impaired fibrinolytic activity, either due to a poor release of t-PA after venous occlusion, or due to increased PAI-1 levels. In two prospective studies carried out on postoperative DVT in subjects subjected to hip replacement, preoperative values of increased PAI-1 appeared to be predictive of postoperative venous occlusion.
Studies regarding a reduced t-PA activity and elevated PAI-1 levels as a risk factor for thromboembolic disease remain to be further evaluated. Elevations of t-PA antigen have been linked to persons at risk in several studies involving cardiovascular events in subjects with angina pectoris and coronary artery stenosis, myocardial infarction MI , and stroke.
Furthermore, a strong support for the link between PAI-1 elevation and risk of having a MI was obtained from a study of men who had survived a first MI before the age of 45 years. Reduced t-PA activity has been reported as predictive for MI, for MI in subjects with angina pectoris, and in ischaemic disease in younger men.
The results from these studies put together suggest that a state of elevated t-PA and PAI-1 antigen and reduced activity is the condition associated with cardiovascular disease. The production of t-PA and u-PA by leukemic cells can be used to predict the prognosis and response to chemotherapy in subjects with acute myeloid leukemia.
Subjects whose cells produce only t-PA have a lower chance of survival and fail to respond to chemotherapy. In contrast, subjects with u-PA producing cancer cells have a higher chance of survival and a better response to chemotherapy. Cardiovascular diseases, such as acute myocardial infarction, stroke, and venous thromboembolism, are probably the major cause of death and disability in an adult population.
The immediate underlying etiology in these conditions is often a thrombotic obstruction of critically situated blood vessels, causing a loss of blood flow to vital organs. One approach to the treatment of thrombosis consists of the intravenous infusion of plasminogen activators as clot-dissolving drugs. The use of thrombolytic agents may occasionally require close monitoring of the components of the plasminogen activation system.
Excessive thrombolytic activity is likely to cause bleeding, particulary cerebral hemorrhage, as a side-effect. An intriguing feature of the fibrinolytic system is the circadian variation in t-PA and PAI-1 that has been observed.
Free t-PA levels are lowest in the morning, increase during the day and reach their peak activity level in the late afternoon. It has been suggested that the high incidence of myocardial infarction and cerebral thrombosis in the morning hours, may be connected to the circadian rhythm of fibrinolytic activity. From mortality statistics in Greenland it is known that Eskimos have a low prevalence of myocardial infarction.
This has been related to their diet, although it may also be due to the observation that Eskimos have a rapid increase in t-PA activity in the morning and a more rapid decrease in PAI-1 activity and antigen compared to Caucasians. Because of the diurnal variation in fibrinolytic activity, sampling should always take place at the same time during the day usually between 8 a. Different stimuli, drugs, and environmental factors have been reported to modulate the fibrinolytic activity in the experimental animal as well as in humans.
Examples of these are listed below in alphabetical order. Most reports on alcohol and fibrinolysis show an increase in plasma PAI-1 levels following alcohol consumption that causes an acute decrease in t-PA activity. In a recent study of moderate alcohol consumption in a group of healthy men, it was observed that t-PA activity falls sharply after alcohol intake for the first 5 hours, although it then rises and becomes significantly higher after 13 hours.
Stanozolol produces profound change in the coagulation and fibrinolytic systems after prolonged oral administration. Peak t-PA concentrations are seen after 40 to 60 minutes of administration.
A rise in the fibrinolytic activity after exercise has been reported by many authors and attributed mainly to the acute release of t-PA from the vascular endothelium. The increase in t-PA activity is related to both the intensity and the duration of exercise and may reach 30 times the normal after a Marathon race.
When comparing physically active and inactive men, it was found that t-PA activity increases more in active men and that they have a lower PAI-1 activity. A diet rich in high-complex carbohydrates and low in fat has been reported to lower both t-PA and PAI-1 antigen. The net effect was an enhancement of fibrinolytic potential, due to the greater fall in PAI When comparing fish with lean meat, it has been observed that a fish diet leads to higher t-PA and PAI-1 antigen levels.
Basal t-PA activity may increase following a 24 hour fast. This is probably a secondary reaction due to decreased PAI-1 activity. Heparin can form complexes with t-PA. The complex has higher catalytic activity for plasminogen activation than t-PA alone, but heparin appears to inhibit the potentiating effect of fibrin on t-PA-induced plasminogen activation.
Prolonged administration of unfractionated heparin and LMW heparin, induces a rise of circulating t-PA antigen levels. The release of t-PA from the endothelium may be involved in the pathogenesis of anaphylactic shock induced by insect venom.
Levels have been found to increase about fold following a controlled insect-sting challenge in subjects with a previous history of insect-sting induced anaphylactic reaction.
Mental stress releases t-PA 54 in a similar manner to that of adrenaline, 55 with increases in heart rate and systolic and diastolic blood pressures. Stress-induced release must be avoided during blood sample collection.
The subject must rest both mentally and physically for minutes prior to venepuncture. Oral contraceptives may produce a significant increase in t-PA activity, not due to decreases in PAI-1 or plasminogen concentration.
Pregnancy induces marked changes in the coagulation mechanism and fibrinolytic system, changes that aim to secure hemostasis during pregnancy and delivery. The increase of t-PA antigen and the decrease of PAI-1 antigen and activity after the 38th week contributes to sustain a fibrinolytic potential capable of degrading large fibrin deposists. In addition, the release of t-PA after a venous occlusion is impaired in chronic smokers.
The venous occlusion test is often used to test subjects for their capacity to release t-PA from the occluded venous segment. Particularly sensitive to mutation are highly conserved amino acids.
Heavy chain deletion variants were constructed from point mutants at the domain boundaries of tPA. Deletion of the kringle domains lowers the fibrinolytic activity to a greater extent than deletion of the finger or growth factor domains.
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