| Cloning
and Expression of RSP12, a Peptidyl-Prolyl Isomerase
Susan
R. Hupp
Wheeling
Jesuit University
Wheeling,
WV
Mentor:
Pinfen Yang
The
radial spoke of Chlamydomonas reinhardtti is an axonemal structure essential
for flagellar motility. The major challenges are to elucidate the assembly
and functional mechanism of the symmetric T-shaped complex consisting of
more than 22 molecules. One of the approaches is to use mass spectrometry
to identify the unknown radial spoke proteins (RSP). RSP12 is a 20 kDA
protein located in the stalk of the T-shaped radial spoke in Chlamydomonas
flagella. Mass spectrometry of RSP12 spot-purified from 2-D gels of isolated
radial spokes revealed the corresponding complete EST and genomic sequences.
Further analysis with EST sequences revealed two potential alternative
spliced forms of the protein. RT-PCR confirmed both forms using message
specific primers. Only one of the two forms, most likely encoding RSP12,
contains all 5 of the peptide sequences derived from mass spectrometry.
BLAST
searches using the predicted protein sequence of RSP12, showed that it
is a homolog of peptidyl-prolyl isomerase (ppI). The function of ppI is
to change conformation of proline residues in polypeptides by converting
them from the cis to the trans form. Members of the ppI family are thought
to play a role in the assembly of molecular complexes. We propose that
RSP12 operates in joining the two “7” shaped arms of the assembling radial
spoke into the “T” shape of a complete radial spoke.
To
test the ppI function of RSP12, RT-PCR was used to obtain the full length
coding sequence, which was subsequently cloned to the PET28a expression
vector. The vector was transformed to BL21 cells for expression. Induction
with IPTG confirms that the protein is present and can be over expressed
in the cells. However, at this point, the protein is precipitating into
pellet and will require purification. |
