In vitro human cell-free expression system for synthesis of malaria proteins.
In this study, we performed cell-free expression of Plasmodium proteins using the in vitro human cell-free protein expression systems for DNA and mRNA. Malaria rhoptry genes (PFc14_0344, PFc0120w, PY01759, PY00763, PY07482, and PY04666) and a Maurer's cleft gene (PfA0680c) identified from proteome analysis studies were cloned into the pT7CFE1-CHis expression vector. Following a coupled transcription-translation procedure, expressed proteins were analyzed by His-tag staining and by western blotting using protein specific antibodies and nickel-horseradish peroxidase (HRP) for histidine detection. Antibodies against whole rhoptries of Plasmodium falciparum and Plasmodium yoelii merozoites and antibodies specific for the PfMC-2TM protein identified translated proteins. The rhoptry specific antibodies exhibited cross reactivity among the expressed proteins of P. falciparum and P. yoelii. The results demonstrate that the in vitro human cell-free protein expression system is suitable for rapid expression and screening of malaria vaccine candidates and diagnostic biomarkers.
Department of Biological, Geological, and Environmental Sciences, College of Sciences and Health Professions, Cleveland State University, Cleveland, OH 44115, USA.
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Yadavalli
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Ledger
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Sam-Yellowe
D015153:Blotting, Western; D002474:Cell-Free System; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010963:Plasmodium falciparum / Q000235:genetics*; D010967:Plasmodium yoelii / Q000235:genetics*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013194:Staining and Labeling
D015800:Protozoan Proteins; D011994:Recombinant Proteins