Constitutive Expression and Optimization of Nutrients for Streptokinase Production by Pichia pastoris Using Statistical Methods

Abstract

The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett-Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.

Optimization of amorphadiene production in engineered yeast by response surface methodology

“…Response surface methodology (RSM) has been widely used to evaluate and understand the interactions between different process parameters (Khuri et al 1987). RSM was applied successfully for optimizing process parameters for various processes in biotechnology, from biological treatment of toxic wastes (Ravichandra et al 2008a, b) to enzyme production (Doddapaneni et al 2007; Tatineni et al 2007; Ravichandra et al 2008a, b; Chennupati et al 2009) including recombinant products (Vellanki et al 2009; Farhat-Khemakhem et al 2012). Till date, studies with statistical optimization of parameters for production of amorphadiene have not been reported elsewhere.…”

Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli

“…Cleavage of plasminogen in zymogen form at an Arg-Val bond generates plasmin, an active enzyme that degrades fibrin component of thrombin [1]. Due to this property, the streptokinase has been widely used in the therapy of acute myocardial infarction for its strong activity in dissolving blood clots [2].…”

“…However, due to low SK production yields from natural host and its pathogenicity, so research interest has shifted to cloning and expression of SK in hyperproductive and safe heterologous host systems. Therefore, sk genes have been cloned and expressed in different expression systems including Bacillus subtilis [3], Streptococcus sanguis [4], Streptomyces lividans [5, 6], Schizosaccharomyces pombe [7], Pichia pastoris [1, 8], Lactococcus lactis [9], and Escherichia coli [10, 11]. However, there are some disadvantages of producing recombinant proteins in Pichia pastoris due to high glycosylation level [12] or in Lactococcus lactis due to low cell density [9].…”

Constitutive Optimized Production of Streptokinase inSaccharomyces cerevisiaeUtilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter ofPichia pastoris

“…The skc gene encoding SK (47 kDa) is produced in a heterologous host that is generally regarded as safe (GRAS) organism. A variety of bacterial and yeast hosts were successfully exploited for the production of active SK [7, 8]. S. cerevisiae , due to its extensive data on gene manipulation tools, is the traditional host for the production of heterologous proteins.…”