Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs.
Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.
Ghosh
S
S
Department of Biochemistry, Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, India.
Ghosh
R
R
Das
P
P
Chattopadhyay
D
D
D000818:Animals; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D016829:Giardia lamblia / Q000235:genetics / Q000502:physiology*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D018076:DNA, Complementary; D016053:RNA, Protozoan; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C047458:fibrillarin