High level production of active streptokinase in Pichia pastoris fed-batch culture
Introduction
Circulatory disorders like ischaemic stroke, acute myocardial infarction, pulmonary embolism and deep-vein thrombosis develop due to the formation of a blood flow impeding clot in the vessels. One of the major treatment options for such cases is the intravenous administration of thrombolytics [1], [2]. Streptokinase (SK) has emerged as a valuable biomolecule for thrombolytic therapy due to its low cost and longer in vivo half-life than other molecules such as urokinase (UK) and tissue plasminogen activator (tPA) [2], [3]. Streptokinase is a 47 kDa (414 amino acids) single chain polypeptide produced extracellularly as a catabolic byproduct by various strains of hemolytic streptococci [4]. It does not possess any enzymatic activity of its own but a 1:1 stoichiometric complex of streptokinase and plasminogen has a trypsin-like serine protease activity involved in dissolution of the fibrin blood clot [3].
The streptokinase gene from Streptococcus equisimilis strain H46A has been cloned and expressed in several heterologous hosts due to the pathogenicity of its natural isolate. In Escherichia coli shake flask culture, the streptokinase expression has been reported in the range of 100 U/mL to 5000 IU/mL [5], [6], [7]. The large scale fed-batch production of streptokinase using E. coli has been reported with improved expression yields up to 1120 mg/L [8], [9], [10], [11]. Yazdani and Mukherjee [12] obtained ∼20,000 IU/mL of streptokinase in continuous culture studies of E. coli. The Challis strain of Streptococcus sanguis secreted ∼1500 U/mL of recombinant streptokinase in large scale fermentation experiments while Proteus mirabilis produced 24,000 U/mL of streptokinase in batch fermentation [13], [14]. However, the recombinant streptokinase production in E. coli has been reported to be associated with problems of high plasmid instability rates and loss of cell viability due to its toxicity towards the expression host [9], [12]. The expression of this toxic protein has been shown to have an adverse effect on cell biomass thereby reducing the final expression yields whereas inclusion body formation inside the cell makes downstream processing very costly [8], [9], [15].
To circumvent these problems, different yeast systems have been used for the expression of this thrombolytic protein. In Pichia pastoris expression system, the gene of interest can be stably integrated into the genome of the expression host to overcome the problem of plasmid instability [16]. Being a eukaryotic host, it carries out post-translational modifications and has high amenability to high cell density fermentation [16], [17]. Another major reason for popularity of this expression host is the presence of efficient and tightly regulated strong promoters such as alcohol oxidase 1 (AOX1) and glyceraldehyde 3-phosphate dehydrogenase (GAP) with highly specific secretion efficacy for recombinant proteins to make downstream processing simple and cost effective [16], [17]. The secretion of glycosylated streptokinase has been reported in P. pastoris (3200 IU/mL) with an improved proteolytic resistance and stability [18]. A mutant of Schizosaccharomyces pombe defective in extracellular protease activity produced streptokinase without any glycosylation and degradation at a level of 4133 IU/mL [19]. However, these expression levels were not satisfactory for commercial production of this immensely important therapeutic molecule.
Therefore in this work, we report the development of a highly efficient recombinant Pichia system for the secretory expression of streptokinase at large scale. Host-vector combinations were optimized at shake flask level to screen a hyper producer strain along with its purification from culture supernatant. Biophysical characterization of purified protein was done using far-UV CD and fluorescence spectroscopy. This is the first report of a high level production of bioactive streptokinase in P. pastoris at bioreactor level where 4.25 g/L of streptokinase was achieved with a volumetric productivity of 3727 IU/mL/h i.e. 57.43 mg/L/h.
Section snippets
Host strains, plasmids and chemicals
E. coli DH5α strain from Amersham Biosciences (USA) was used for cloning and plasmid propagation. Plasmids were isolated by standard alkaline lysis method. All media constituents were purchased from HiMedia (India) and chemical reagents were from Fisher Scientific (USA). Low salt LB medium was used to grow E. coli cultures. Agar was added at a concentration of 2% to make agar media plates. The P. pastoris expression system including the host strain X-33 and GS115 (his4) and the expression
Fermentation studies
The fermentation studies were conducted in a 2.5 LElectrolab (FerMac 310/60) bioreactor (UK) using computer controlled Electrolab Fermentation Manager SCADA software at 1 L working volume. The batch media used for fermentation studies consisted of 2% yeast extract, 4% peptone and 5% dextrose, 100 mM potassium phosphate buffer (pH 6.0) and 0.67% YNB. The culture pH was maintained at 6.0 using 28% liquor ammonia and 50% orthophosphoric acid. Primary inoculum was prepared in YPD broth while the
Construction, transformation and screening of recombinant Pichia clones
Streptokinase is a key biological macromolecule involved in blood clot dissolution. The mature streptokinase gene of 1242 bp was cloned under AOX1 and GAP promoter having N-terminus α-mating factor signal sequence for its extracellular expression. Constructs having C-terminus 6xHis tag were also generated to facilitate downstream processing of the recombinant streptokinase. The schematic representation of all the constructs has been given in Fig. 1. The streptokinase protein without tag is
Discussion
Cardiovascular diseases (CVDs) are one of the biggest global killers in the present health scenario where streptokinase can be a molecule with immense therapeutic value to the developing countries [1], [2]. It has been produced in several heterologous hosts with varying degrees of success [5], [13], [14], [18], [19], [26]. Therefore, we have attempted to resolve the problem of its poor expression in eukaryotic hosts via host-vector combination followed by bioprocess optimization. The inaccurate
Conclusions
The recombinant proteins’ toxicity towards expression host remains a significant deterrent for their large scale production. In this study, the expression of recombinant SK, which is toxic to its host, was enhanced many folds by the simple expedient of getting the product in the culture supernatant. The highest product accumulation in culture supernatant was 4.25 g/L in high cell density fermentation. The high purity and expression yield achieved for this therapeutically important macromolecule
Authors’ contribution
Adivitiya participated in all the experiments and executed cloning, shake flask optimization and biophysical characterization work with manuscript writing. VKD worked on bioprocess optimization work and ND helped in carrying out the activity assay. YPK conceived the study and participated in drafting and editing of the manuscript. All authors read and approved the final manuscript without any conflict of interests.
Acknowledgements
This work was financially supported by R&D grant of University of Delhi. Adivitiya, Vikas Kumar Dagar, and Nirmala Devi are the recipients of senior research fellowship from Council of Scientific and Industrial Research (CSIR), Indian Council of Medical Research (ICMR), and University Grants Commission (UGC), Govt. of India, New Delhi, respectively.
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