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Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris

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Abstract

Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1–A2) and light chains (A3–C1–C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.

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Abbreviations

AOX:

Alcohol oxidase

DAB:

3, 3'-diaminobenzidine

ELISA:

Enzyme-linked immunosorbent assay

HRP:

Horseradish peroxidase

PCR:

Polymerase chain reaction

YPD:

Yeast extract peptone–dextrose

YPDS:

Yeast extract peptone–dextrose–sucrose

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Acknowledgments

The authors would like to thank Dr. Rajan Dighe, IISc, Bangalore, for providing the host strain Pichia pastoris X-33, vector pPICZα-A and Dr. Alok Srivastava, CMC Hospital, Vellore, for BDD factor VIII cDNA. Thanks to Ms. Revathi E M for providing the anti-C2 polyclonal antibody. The financial support from DBT is gratefully acknowledged.

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  1. Center for Bio Separation Technology (CBST), VIT University, Vellore, 632014, Tamil Nadu, India

    Sudheer Reddy A.R., Padikara Kutty Satheeshkumar & Mookambeswaran A. Vijayalakshmi

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  1. Sudheer Reddy A.R.
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  2. Padikara Kutty Satheeshkumar
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  3. Mookambeswaran A. Vijayalakshmi
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Correspondence to Mookambeswaran A. Vijayalakshmi.

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A.R., S.R., Satheeshkumar, P.K. & Vijayalakshmi, M.A. Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris . Appl Biochem Biotechnol 171, 10–19 (2013). https://doi.org/10.1007/s12010-013-0338-4

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