Recombinant Streptokinase Research Articles

Platelet Membrane-Coated r-SAK Improves Thrombolytic Efficacy by Targeting Thrombus.

Thrombolytic therapy is one of the most effective treatments for thrombus dissolution and recanalization of blocked vessels in thrombotic diseases. However, the application of the thrombolytic strategy has been limited due to unsatisfactory thrombolytic efficacy, relatively higher bleeding complications, and consequently restricted indications. Recombinant staphylokinase (r-SAK) is a third-generation thrombolytic agent produced by genetic engineering technology, which exhibits a better thrombolytic efficacy than urokinase and recombinant streptokinase. Inspired by the natural affinity of platelets in hemostasis and pathological thrombosis, we developed a platelet membrane (PM)-coated r-SAK (PM-r-SAK). Results from animal experiments and human in vitro studies showed that the PM-r-SAK had a thrombolytic efficacy equal to or better than its 4-fold dose of r-SAK. In a totally occluded rabbit femoral artery thrombosis model, the PM-r-SAK significantly shortened the initial recanalization time compared to the same dose and 4-fold dose of r-SAK. Regarding the recanalized vessels, the PM-r-SAK prolonged the time of reperfusion compared to the same dose and 4-fold dose of r-SAK, though the differences were not significant. An in vitro thrombolytic experiment demonstrated that the thrombolytic efficacy of PM-r-SAK could be inhibited by platelet-poor plasma from patients taking aspirin and ticagrelor. PM coating significantly improves the thrombolytic efficacy of r-SAK, which is related to the thrombus-targeting activity of the PM-r-SAK and can be inhibited by aspirin- and ticagrelor-treated plasma.

Biotechnological Advancements in Streptokinase Production

This article offers a deep analysis of activating streptokinase which looks into the biotechnological challenges and future perspectives. One of the main problems associated with streptokinase is its short half-life, complex production process, and immunogenicity which hinders patients from getting a successful reperfusion. Yet, the development of biotechnology methods that would not face these drawbacks, such as the strategy of recombinant streptokinase production and enzyme engineering theories, is a promise. This paper aims to provide an overview of biotechnological breakthroughs in the production of streptokinase with a focus on discovering long-lasting formulations exhibiting less immunogenicity and in addition the economic viability and scalability of biotechnological production processes. This article also gives the comping image of known purification methods like chromatography, ultrafiltration, and I-A chromatography for the separation and purification of streptokinase. In addition, the author presents the advantage of such suitable production technologies as cell-free production systems and E. Coli system optimization, such as recombinant streptokinase synthesis in Yeast systems. Biotechnology is turning out to be a new area, having a future boom due to the rising innovation and demand that are associated with the prospects revealed by the market trends.

Evaluating the thrombolytic property of Juniperus Sabina tincture using spectrophotometry

Objectives: We aimed to explore the thrombolytic property of Juniperus Sabina by measuring the absorbance level of released haemoglobin (Hb) during clot lysis. Materials and Methods: Approximately 0.3 mL of blood was added to four 15 mL centrifuge tubes and blood clots were allowed to form naturally. The clots were incubated for 45 min and washed with normal saline. Using a visible spectrophotometer, the absorbance of Hb was recorded at five wavelengths: 405, 416, 541, 576 and 580 nm. Lyophilised recombinant streptokinase (SK) was used as standard control. J. Sabina mother tincture (83.28% v/v) of 1:10 (T1) and 1:20 (T2) dilutions were studied. Distilled water and ethyl alcohol mixed in a ratio of 1:1 (hydro alcohol) were used as a negative control. Results: Thrombolytic activity was compared by recording the absorbance of Hb at five wavelengths (405, 416, 541, 576 and 580 nm) every 10 min from 0 to 60 min. Absorption of light by the released Hb during thrombolysis was highest at 416 nm for SK, T1 at 405 and 416 nm and HA and T2 at 405 nm. Comparatively, T1 showed higher absorption than HA. In general, the peak absorbance of Sabina concentrations was more when compared to negative controls in the higher wavelengths of 541, 576 and 580 nm. However, this difference was not statistically significant. The mean and standard deviation from the data were calculated to find the amount of dispersion for all the samples at different wavelengths. The within- and between-group significance was determined using analysis of variance. Conclusion: This study has shown that Sabina in the dilutions of 1:10 and 1:20 does not induce thrombolysis in vitro. By and large, further studies with different concentrations of Sabina Q with a sophisticated spectrophotometer need to be tested for thrombolytic activity.

Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity

Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity . Synthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard. Seed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x10 6 IU/mg with 62.94 ± 2.3% clot lysis efficiency. A high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.

Evaluation and comparison of affinity chromatography and precipitation– based methods on purification of recombinant streptokinase

Evaluation and comparison of affinity chromatography and precipitation– based methods on purification of recombinant streptokinase

Thrombolytic effects of recombinant staphylokinase on coronary thrombosis in miniature pigs

To evaluate the thrombolytic effects of recombinant staphylokinase and compare it with those of recombinant streptokinase. Thirty Chinese experimental miniature pigs were divided into five groups, namely, solvent control group, positive drug control group and three recombinant staphylokinase groups, six in each group. The thrombus of coronary artery was formed by surgical thoracotomy and direct current stimulation in anesthetized animals. Intravenous administration was started after the thrombus of coronary artery was formed for 30 minutes, and the method of first injection and then constant speed infusion by peristaltic pump was used. The solvent control group was injected intravenously with solvent, the positive drug control group was given recombinant streptokinase 4 mg·kg-1 intravenously, and the three recombinant staphylokinase groups were given recombinant staphylokinase at the doses of 4, 2 and 1 mg.kg-1 intravenously. The volume of intravenous injection was 5 ml, which was completed within 1 min, the speed of infusion was 0.5 ml·min-1, which was completed within 60 min, and the animals were sacrificed 120 minutes later. Before and 30, 60 and 120 min after administration, the venous blood samples were collected. At the end of the experiment, the coronary artery segments of the thrombosis site were taken, and the euglobulin dissolution time (ELT), blood fibrinogen content (FBG), fibrinogen degradation product (FDP) and wound bleeding volume were measured respectively. The coronary thrombolysis rate, myocardial ischemia degree and ischemia range were measured. Compared with the solvent control group, ELT in the experimental group was significantly shortened (P＜0.05 or P＜0.01), FBG degradation in a few experimental animals was more than 20%, FDP was significantly increased (P＜0.05 or P＜0.01), and there was no significant effect on blood pressure and heart rate of small pigs. Compared with the control group, the maximum thrombus area was decreased by 34.3% and 15.4% (P＜0.05) in the high and middle dose groups of the experimental group. Compared with the same dose of recombinant streptokinase, recombinant staphylokinase had stronger thrombolytic effect (P＜0.05 or P＜0.01) on the coronary thrombus caused by electrical stimulation, less bleeding side effects and the same effect on the degree and range of myocardial infarction as recombinant streptokinase. Compared with recombinant streptokinase, recombinant staphylokinase has faster thrombolysis speed, higher fibrin specificity and less bleeding side effects. In general, 2 mg.kg-1 recombinant staphylokinase has better efficacy and safety.

Investigating the functional relationship between streptokinase variants from Group A Streptococcus, and associated M-like proteins

Background: Streptokinase (SK) from Group A streptococcus (GAS) activates human plasminogen to generate plasmin, which degrades fibrin clots tofacilitate bacterial dissemination. Sequence variants of SK from diverse GAS strains form distinct evolutionary clusters.Unlike cluster 1 SK, cluster 2 variants have very little activity in solution and depend on co-factors (e.g. fibrin(ogen)).Cluster 2 SK variants also appear to correlate with cell-surface M-like proteins: SK2a with M1 (fibrinogen-binding) and SK2b with PAM (plasminogen-binding). Methods: Plasminogen activation by recombinant SKs (rSK2a, rSK2b) was investigated by chromogenic assay; nickel-coated microtiter plates were used to immobilise recombinant M proteins (rPAM and rM1) via a C–terminal His tag to mimic cell surface plasmin generation. Results: Plasminogen activation by rSK2b is stimulated ∼18-fold by rPAM in solution; when rPAM is immobilised stimulation exceeds 100-fold. Fibrin is the most potent stimulator of rSK2a activity (7-fold increase) compared to fibrinogen (4-fold); when rM1 was included, either in solution or immobilised, there was no further stimulation of rSK2a activity with fibrinogen. Discussion: Stimulation ofSK2b activity by plasminogen bound to immobilised PAM suggests an important role for cell-surface plasmin generation. SK2a activity appears to be independent of M1, targeting fibrin directly. SK variants are commonly associated with distinct disease manifestations with SK2b commonly expressed by invasive skin-tropic strains of GAS and SK2a by nasopharynx-tropic strains. An improved understanding of the molecular mechanism of action by GAS SK variants may help to identify potential novel therapeutic targets for the treatment of invasive GAS diseases.

Development of a streptokinase expression platform using the native signal sequence of the protein with internal repeats 1 (PIR1) in P. pastoris: Gene dosage optimization and cell retention strategies

Abstract Streptokinase is a therapeutically important thrombolytic agent which is toxic to its heterologous expression hosts and also prone to proteolytic degradation. In the current study, its secretory expression was targeted in P. pastoris using the native protein with internal repeats 1 (PIR1) secretion signal where the optimization of streptokinase gene dosage resulted in an increase in the volumetric product yield from 339.69 to 1206.48 mg/L. The cell retention strategy was applied during the induction phase to maintain sustained productivity where up to a 2.4 fold enhancement in the overall product yield was achieved at shake flask to a level of 3049.53 mg/L. Moreover, the cell recycling approach prevented the streptokinase proteolytic degradation and also helped in achieving a high specific product yield (YP/X) in a range of 167.65–176.44 mg/g DCW even after five cycles. Similarly, the cell recycling strategy was also employed using S. cerevisiae α-mating factor (α-MF) signal sequence which also resulted in improved product stability and volumetric product concentration confirming its wide applicability in obtaining enhanced product quality and yield. The recombinant streptokinase produced using PIR1 signal sequence was biologically active possessing a specific activity of 62,782.40 IU/mg and was predicted to contain 10.45% α-helix and 29.09% β-sheet.

Purification and characterization of recombinant Streptokinase expressed in E.coli from Streptococcus equisimilis with N-terminal methionine

Streptokinase is a extracellular enzyme which is extracted from strains of beta Hemolytic streptococcus. The enzyme is a non-protease plasminogen activator that activates plasminogen to plasmin and degrades fibrin clot through its specific lysine binding site which is used in thrombolytic therapy. Purification of streptokinase produced from S.equisimilis in E.coli with N-terminal methionine was carried out in 3 Chromatography purification steps, 1) CM-Sepharose-FF at pH 4.2 followed by concentration and dialysis over night with Tris-HCl pH 8.0. Partially purified dialyzed enzyme sample was loaded on to 2) DEAE-Sepharose-FF column. The Purified fractions of DEAE column were pooled and applied on to Sephadex G-100 column. Enzyme purity was confirmed by SDS-PAGE and RP-HPLC.Its biological activity is determined by specific streptokinase assay and characterised the enzyme by Peptide mapping, MALDI-TOF, Isoelectric-focusing and RP-HPLC. The isoelectric point (pI) of streptokinase is around 4.98.The results of characterization shows that it contains two forms (Isomers) of streptokinase expressed in E. coli which was analyzed by RP-HPLC and chromogenic assay. The variation is formed by isomer-1 in which 85% of Streptokinase expressed without methionine (85000IU/mg) and Isomer-2 in which 15% of streptokinase expressed with methionine (nil activity) in E. coli. This phenomenon shows that the presence and absence of methionine in isomers of streptokinase varying the catalytic activity of the enzyme.

Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris.

Streptokinase, a therapeutically important thrombolytic agent, is prone to C-terminal degradation and plasmin-mediated proteolytic processing. Since the protein was glycosylated during secretion from Pichia pastoris, therefore, the role of carbohydrate moieties on its stability was analyzed via in vivo blocking of N-glycosylation using tunicamycin where an increased degradation of streptokinase was observed. Further, the in vitro site-directed mutagenesis of the three putative N-glycosylation sites at asparagine residues 14, 265, and 377 to alanine revealed the essentiality of glycosylation of the 14th amino acid residue in its post-translational proteolytic stability without significantly affecting its biological activity. However, the mutation of both Asn265 and Asn377 did not seem to contribute toward its glycosylation but resulted in a 39% lower specific activity in case of the rSK-N265,377A. Moreover, the mutation of all three glycosylation positions drastically reduced the secretory expression of native streptokinase from 347 to 186.6mg/L for the triple mutant with a 14% lower specific activity of 56,738IU/mg from 65,808IU/mg. The secondary structure, tertiary structure, and thermal transition point (45-55°C) of all the deglycosylated variants did not show any significant differences when compared with fully glycosylated native streptokinase using CD and fluorescence spectroscopy. Furthermore, the longer acting plasmin-resistant variants were also developed via the mutation of lysine residues 59 and 386 to glutamine which enhanced its biological stability as a ~ 1.5-fold increase in the caseinolytic zone size was observed in case of rSK-K59Q and also in rSK-K59,386Q mutant without affecting the structural properties.

Structural characterization of recombinant streptokinase following recovery from inclusion bodies using different chemical solubilization treatments

Circular dichroism (CD) in far-UV region was employed to study the extent of changes occurred in the secondary structures of recombinant streptokinase (rSK), solubilized from inclusion bodies (IBs) by different chemicals and refolded/purified by chromatographic techniques. The secondary structure distribution of rSK, obtained following different chemical solubilization systems, was varied and values in the range of 12.4–14.5% α-helices, 40–51% β-sheets and 35.5–48.3% turns plus residual structures were found. With reducing the concentration of chemicals during IB solubilization, the content of turns plus random coils was diminished and simultaneously the amounts of α- and β-sheets were increased. These changes in the secondary structures would lower the hydrophobicity along with the chance of protein aggregation and expose the hydrophilic regions of the protein. Therefore, these alterations in the secondary structures, occurred following efficient IBs solubilization by low concentration of chemicals, could be related to enhancement in rSK biological potency previously observed.

Solubilization of inclusion body proteins using low and very low concentrations of chemicals: implications of novel combined chemical treatment designs in enhancement of post‐solubilization target protein purity and biological activity

AbstractBACKGROUNDSolubilization of inclusion body (IB) proteins by conventional methods (i.e. high concentrations of denaturants such as chaotropes) is a challenging process due to denaturation of the native‐like secondary structures and subsequently low recovery into bioactive forms. The main objective of this work was to study the effect of a range of chemicals at low and very low concentrations on the solubilization of IB proteins produced in Escherichia coli and the enhancement of the target protein biological activity subsequent to refolding.RESULTSPerformance of chemical combinations at low and very low concentrations through the solubilization process of recombinant streptokinase (rSK) from IBs isolated from E. coli was appraised based on values pertinent to three parameters including target protein solubilization yield, purity and biological activity. In comparison with the conventional IBs' solubilization method (i.e. 4 mol L‐1 urea), combinations of 0.5–1 mol L‐1 urea with very low to low concentrations (0.05–1%) of detergents resulted in considerable target protein solubilization (by 100%), very high post‐solubilization target protein purities (up to 100%) and biological activities (up by 360%).CONCLUSIONOwing to the improvements in the abovementioned integral parameters, these chemical treatments are good candidates to be considered for more efficient and cost‐effective recovery of recombinant target proteins from IBs. © 2017 Society of Chemical Industry

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product

ABSTRACTRecombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli’s and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (101 to 107 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Cell‐free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care.

Sequence and kinetic analyses of streptokinase from two group G streptococci with high fibrin-dependent plasminogen activities and the identification of novel altered amino acids as potential hot spots.

To gain insights on the degree of heterogeneity and kinetic differences of streptokinase (SK) from group G (SKG) Streptococci compared with standard SK from group C (SKC) and identification of potentially contributing critical residues (hotspots). DNA and sequencing analyses confirmed the proper construction of all SK encoding vectors (two SKGs and one standard SKC). SDS-PAGE and western blot analyses confirmed the expression and proper purification of the recombinant SKs from E.coli with the expected size of 47kDa. Kinetic analyses of two SKGs, compared with SKC, showed higher levels of specific [(×103IU/mg) of 725 and 715 vs. 536] and fibrin-dependent proteolytic activities [Kcat/KM (min-1/µM) of 37 and 30 vs. 23], accompanied by declined fibrin-independent amidolytic activities [Kcat/KM (min-1/mM) of 109 and 84 vs. 113], respectively. Sequence alignments identified 10 novel residual substitutions scattered in SKα (I33F, R45Q, SKG132, A47D, and G55N), SKβ (N228K, F287I), and SKγ domains (L335V, V396A, T403S) of SKGs, as potential hotspots. The residue substitutions identified might critically contribute as hot spots to different kinetic parameters of SKGs and might assist in further elucidation of structure/function relations and rational design of SKs with improved (fibrin-dependent) therapeutic properties.

Activity Regulation by Fibrinogen and Fibrin of Streptokinase from Streptococcus Pyogenes.

Streptokinase is a virulence factor of streptococci and acts as a plasminogen activator to generate the serine protease plasmin which promotes bacterial metastasis. Streptokinase isolated from group C streptococci has been used therapeutically as a thrombolytic agent for many years and its mechanism of action has been extensively studied. However, group A streptococci are associated with invasive and potentially fatal infections, but less detail is available on the mechanism of action of streptokinase from these bacteria. We have expressed recombinant streptokinase from a group C strain to investigate the therapeutic molecule (here termed rSK-H46A) and a molecule isolated from a cluster 2a strain from group A (rSK-M1GAS) which is known to produce the fibrinogen binding, M1 protein, and is associated with life-threatening disease. Detailed enzyme kinetic models have been prepared which show how fibrinogen-streptokinase-plasminogen complexes regulate plasmin generation, and also the effect of fibrin interactions. As is the case with rSK-H46A our data with rSK-M1GAS support a “trigger and bullet” mechanism requiring the initial formation of SK•plasminogen complexes which are replaced by more active SK•plasmin as plasmin becomes available. This model includes the important fibrinogen interactions that stimulate plasmin generation. In a fibrin matrix rSK-M1GAS has a 24 fold higher specific activity than the fibrin-specific thrombolytic agent, tissue plasminogen activator, and 15 fold higher specific activity than rSK-H46A. However, in vivo fibrin specificity would be undermined by fibrinogen stimulation. Given the observed importance of M1 surface receptors or released M1 protein to virulence of cluster 2a strain streptococci, studies on streptokinase activity regulation by fibrin and fibrinogen may provide additional routes to addressing bacterial invasion and infectious diseases.

Cloning, purification and enzymatic assay of streptokinase gene from Streptococcus pyogenes in Escherichia coli

Streptokinase is an important therapeutic enzyme that acts as a fibrinolytic agent and use in the treatment of multi serious and life threatening diseases such as pulmonary embolism and acute myocardial infarction. Cloning and extracellular expression of recombinant streptokinase from Streptococcus pyogenes was done by fusing the gene coding for streptokinase to an efficient vector (pET15b) with NdeI and BamHI as restriction enzymes and T4 DNA ligase. Streptokinase activity was determined using synthetic chromogenic substrate S-2251, and measuring the absorbance at 405 nm in a UV-spectrophotometer; and purification done under N-terminal 6× histidine tag complementation using affinity chromatography. A 1323-bp of the structural streptokinase gene was amplified. The DNA and amino acid sequence alignments resulting from the BLAST search of streptokinase showed high sequence identity with the other strains of S. pyogenes. The recombinant enzyme had the same molecular weight as other reported streptokinase and the E. coli transformants showed high streptokinase activity. Comparative clinical trials and cost-effectiveness considerations between several major available thrombolytic agents suggest that streptokinase is the drug of choice for thrombolytic therapy particularly in the poorer economies.

Exploration of Recombinant Streptokinase Degradation Products Under Various pH Reduction Conditions in Downstream Processing.

Methods of producing streptokinase, which can be used in the treatment of myocardial infarction, by hemolytic streptococci and recombinant E. coli have been described in patents since 1955. Degradation products in active pharmaceutical ingredients (APIs) and finished pharmaceutical products are considered as impurities and it is required that these degradation impurities are minimized or rather avoided throughout manufacturing process. The aim of this study was to explore the occurrence of rSK degradation during acidification step in downstream processing. The polyclonal antibody was produced by immunization of New Zealand white (NZW) rabbit with pure rSK (purity>98%). The solubilized inclusion bodies with various pH values (4.2, 5.0 and 6.0) were analyzed by Western blotting using rSK polyclonal antibody. Western blot analysis demonstrated the generation of rSK degradation products (with the molecular weight of about 27, 20 and 17 kDa) when the pH value of the solubilized inclusion bodies was reduced to 5.0 and 4.2, while no degradation of rSK observed at pH 6.0. This study demonstrates that the level of pH reduction in the solubilized inclusion bodies during downstream processing plays an important role in generating rSK degradation products, and substantial post-solubilization degradation of rSK occurs at pH lower than 6.0. Development of these degradation impurities, which cannot be eliminated by subsequent chromatographic purifications, can be exclusively avoided during acidification procedure by appropriate pH adjustment approach in downstream processing.

High level production of active streptokinase in Pichia pastoris fed-batch culture

Streptokinase is a biological macromolecule involved in dissolution of fibrin blood clot and favourably used in various clinical applications. This protein is poorly expressed in soluble form due to its toxic effects on host physiology. The extracellular expression of recombinant streptokinase (SK) with and without 6xHis tag was obtained by cloning its gene under the α-mating factor signal sequence and alcohol inducible AOX1 promoter. Host-vector combinations were optimized to select a hyper producer. From shake flask optimization studies, a maximum expression of 582mg/L of rSK (non-tagged) and 538mg/L of rSK-His (His-tagged) protein was obtained when cells were induced at OD600 of 20. The high cell density fermentation increased the volumetric product concentration of rSK-His to a level of 4.25g/L with a 7.9 folds increase from shake flask results. The specific product yield (YP/X) was 49.75mg/g DCW along with a high volumetric productivity of 57.43mg/L/h. The protein was predicted to have 15.43% α-helix and 26.43% β-sheet with tryptophan emission maxima of around 347nm. The highest specific activity of rSK-His was 64,903IU/mg with 1.48 folds purification whereas specific activity of rSK was 55,240IU/mg with 1.22 folds purification.

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach.

Background:Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations. Methods: A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry. Results:The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA. Conclusion:Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals.