Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying a hexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5 % of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100 % with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody.

Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro refolding to increase recombinant proteins production. Additives increase protein refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process. In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA) in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and Dithiothreitol (DTT) on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM. Glutathione pairs (GSH-GssG) showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the pres

http://www.ijbiotech.com/article_7140_a7651af40f069fcfee468002418e0520.pdf

Optimizing refolding condition for recombinant tissue plasminogen activator

In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.

Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation.

Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.

High level expression of kringle 5 fragment of plasminogen in Pichia pastoris.

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelial cell specifically, and shows promise in anti‐angiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When previously expressed in Escherichia coli, recombinant kringle 5 mainly deposited as inactive, insoluble inclusion bodies and the refolding yield was low. In the present study, human kringle 5 was fusion‐expressed with GST (gluthathione‐S‐transferase) under the control of T7 promoter in E. coli. The IPTG‐induced GST‐kringle 5 was about 20% of the total cellular proteins and, among the expressed GST‐kringle 5 proteins, 80% was present in the supernatant. The GST‐kringle 5 fusion protein exhibited some anti‐proliferation activity towards bovine capillary endothelial cells. After GST‐kringle 5 purification, subsequent enterokinase release of intact kringle 5 from the ...

Expression of Biologically Active Kringle 5 Domain of Human Plasminogen in Escherichia coli

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When expressed in E. coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low. In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression. On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture. After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture.

Expression of Kringle 5 Domain of Human Plasminogen in Pichia pastoris

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Expression of Kringle 5 Domain of Human Plasminogen in Pichia pastoris

Expression of Biologically Active Kringle 5 Domain of Human Plasminogen in Escherichia coli