title: Expression, purification and characterization of Plasmodium falciparum vacuolar protein sorting 29.
abstract: Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Mohd Shameel
authors.initials: MS
authors.lastname: Iqbal
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Asim Azhar
authors.initials: AA
authors.lastname: Siddiqui
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Athar
authors.initials: A
authors.lastname: Alam
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Manish
authors.initials: M
authors.lastname: Goyal
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Chinmoy
authors.initials: C
authors.lastname: Banerjee
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Souvik
authors.initials: S
authors.lastname: Sarkar
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Somnath
authors.initials: S
authors.lastname: Mazumder
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Rudranil
authors.initials: R
authors.lastname: De
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Shiladitya
authors.initials: S
authors.lastname: Nag
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
authors.forename: Shubhra Jyoti
authors.initials: SJ
authors.lastname: Saha
authors.affiliation: Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India. Electronic address: ubandyo_1964@yahoo.com.
authors.forename: Uday
authors.initials: U
authors.lastname: Bandyopadhyay
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008018:Life Cycle Stages; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000378:metabolism* / Q000502:physiology; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins
keywords: Cloning; Plasmodium falciparum; Protein expression and purification; Subcellular localization ​PfVPS29