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Over Expression, Purification and Crystallization of the PTF1‐J and PTF‐L Complexes
Author(s) -
Kohrs Alix,
Stein Kelly,
Zhang Mengqing,
Shuller Lauren,
Marshall Andrea,
Christy Rachel,
MacDonald Raymond,
Coats Ward
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.678.12
Subject(s) - transcription factor , transcription (linguistics) , biology , microbiology and biotechnology , gene , genetics , philosophy , linguistics
Students in Biomedical Research Studies at Hillcrest High School in Dallas, Texas, participate in a ongoing research program at the University of Texas Southwestern Medical Center to study the structure and function of a mammalian transcription factor complex. Ptf1a is a transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a common E‐box binding protein (E12/47, HEB, or TCF12), Ptf1a and either Rbpj or Rbpjl. The Rbpj form of the complex (PTF1‐J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl‐form (PTF1‐L) is required for the formation of mature acinar cells. PTF1‐L is involved in an auto‐regulatory loop for the maintenance of transcription of both Ptf1a and Rbpjl genes. We have utilized PCR to clone full length and truncated forms of Ptf1a, HEB, E12/47, Rbpj and Rbpjl. These cDNAs have been subcloned into the bacterial expression vector pSMT3. The over expressed proteins will be purified and crystallized in order to perform structural determination of PTF1‐J and PTF1‐L complexes. Determination of the structures for the PTF1‐J and PTF1‐L complexes will elucidate the structural motifs required for the protein‐protein interactions found in the trimeric complex, as well as the mechanism and the structural determinants for the strong cooperative DNA binding of the complexes.