NDLI: Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli

Content Provider	PubMed Central
Author	Dutta, Sheetij Lalitha, P. V. Ware, Lisa A. Barbosa, Arnoldo Moch, J. Kathleen Vassell, Meredith A. Fileta, Bader B. Kitov, Svetlana Kolodny, Nelly Heppner, D. Gray Haynes, J. David Lanar, David E.
Copyright Year	2002
Abstract	The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83Gly to 531Glu) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.
Related Links	http://dx.doi.org/10.1128/IAI.70.6.3101-3110.2002
Starting Page	3101
File Format	PDF
ISSN	10985522
e-ISSN	10985522
Journal	Infection and Immunity
Issue Number	6
Volume Number	70
Language	English
Publisher	American Society for Microbiology
Publisher Date	2002-06-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Parasitology Immunology Microbiology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Purification, Characterization, and Immunogenicity of a Disulfide Cross-Linked Plasmodium vivax Vaccine Candidate Antigen, Merozoite Surface Protein 1, Expressed in Escherichia coli

Production of the Subdomains of the Plasmodium falciparum Apical Membrane Antigen 1 Ectodomain and Analysis of the Immune Response

Functional Analysis of Plasmodium falciparum Apical Membrane Antigen 1 Utilizing Interspecies Domains

Phase 1 randomized controlled trial to evaluate the safety and immunogenicity of recombinant Pichia pastoris-expressed Plasmodium falciparum apical membrane antigen 1 (PfAMA1-FVO [25-545]) in healthy Malian adults in Bandiagara

Biochemical and Immunological Characterization of Bacterially Expressed and Refolded Plasmodium falciparum 42-Kilodalton C-Terminal Merozoite Surface Protein 1

Allele Specificity of Naturally Acquired Antibody Responses against Plasmodium falciparum Apical Membrane Antigen 1

Binding Hot Spot for Invasion Inhibitory Molecules on Plasmodium falciparum Apical Membrane Antigen 1†

In Immunization with Plasmodium falciparum Apical Membrane Antigen 1, the Specificity of Antibodies Depends on the Species Immunized▿ †

Overcoming Allelic Specificity by Immunization with Five Allelic Forms of Plasmodium falciparum Apical Membrane Antigen 1

Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli

Similar Documents

Purification, Characterization, and Immunogenicity of a Disulfide Cross-Linked Plasmodium vivax Vaccine Candidate Antigen, Merozoite Surface Protein 1, Expressed in Escherichia coli

Production of the Subdomains of the Plasmodium falciparum Apical Membrane Antigen 1 Ectodomain and Analysis of the Immune Response

Functional Analysis of Plasmodium falciparum Apical Membrane Antigen 1 Utilizing Interspecies Domains

Phase 1 randomized controlled trial to evaluate the safety and immunogenicity of recombinant Pichia pastoris-expressed Plasmodium falciparum apical membrane antigen 1 (PfAMA1-FVO [25-545]) in healthy Malian adults in Bandiagara

Biochemical and Immunological Characterization of Bacterially Expressed and Refolded Plasmodium falciparum 42-Kilodalton C-Terminal Merozoite Surface Protein 1

Allele Specificity of Naturally Acquired Antibody Responses against Plasmodium falciparum Apical Membrane Antigen 1

Binding Hot Spot for Invasion Inhibitory Molecules on Plasmodium falciparum Apical Membrane Antigen 1†

In Immunization with Plasmodium falciparum Apical Membrane Antigen 1, the Specificity of Antibodies Depends on the Species Immunized▿ †

Overcoming Allelic Specificity by Immunization with Five Allelic Forms of Plasmodium falciparum Apical Membrane Antigen 1

Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli