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Expression of wheat eukaryotic initiation factor (eIF) 3 in E. coli
Author(s) -
Wilkerson Chris,
Cho Peter,
Mu Kathy,
Shie Jessica,
Tzeng Shinetin,
Fontenot Miles,
Koythong Tamisa,
Lay Erica,
Browning Karen S.,
Choy Grace
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.467.10
Subject(s) - biology , protein subunit , plasmid , eif4a1 , eukaryotic translation , ribosome , ribosomal rna , initiation factor , microbiology and biotechnology , genetics , messenger rna , gene , translation (biology) , rna
The eukaryotic initiation factor 3 (eIF3) complex is comprised of 13 non‐identical subunits. eIF3 interacts with several other initiation factors and controls the assembly of the 40S ribosomal subunit on mRNA. It would be useful to express the subunits for eIF3 in E. coli and purify a functional complex for further analysis. A Freshman Research Initiative (FRI) group prepared de novo all the subunits for wheat eIF3 in a format that optimized codon usage for E. coli. The goal was to clone all 13 eIF3 subunits into three plasmid cassettes for simultaneous expression and self‐assembly into the functional eIF3 complex. The FRI has students from the spring of the freshman year to the fall of the sophomore years participate in goal oriented research. Over a period of 2 years, FRI students prepared coding regions by overlap extension PCR of oligonucleotides, cloned the PCR products into vectors and showed expression of the individual subunits in E. coli. The 13 subunits were cloned into one of three plasmids, pET22b (subunits i,g,h,f), pRSFDuet‐1 (subunits c,d,l,k,e), and pACYCDuet‐1 (a,b, m). Each of the subunits in the plasmids were shown to express. Additional orientations will be tested for optimized expression of the complex. Grant Funding Source : NSF