Premium
Heterologous Expression of the Human Polybromo‐1 Protein in the Methylotrophic Yeast Pichia pastoris
Author(s) -
Hopson Sarah,
Thompson Martin
Publication year - 2016
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.30.1_supplement.579.1
Subject(s) - bromodomain , histone , biology , nucleosome , chromatin , acetylation , high mobility group , microbiology and biotechnology , dna , genetics , gene
The human polybromo‐1 protein (BAF180) is known to be a driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. In its native form, BAF180 is the chromatin‐targeting subunit of the PBAF complex. The precise interactions of BAF180 with the nucleosome are unknown because the recombinant full‐length BAF180 protein has not been available for investigations; only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. BAF180 (encoded by the PBRM1 gene) has nine domains: six bromodomains, two BAH (bromo‐adjacent homology) domains, and one HMG (high mobility group) box. Bromodomains are known to recognize acetylated‐lysines on histones; it is believed that these domains play a crucial role in nucleosome recognition. The BAH domains are critical for the ubiquitination of PCNA, a key regulator of DNA damage. The HMG box is known to be a DNA‐binding domain. Most often, the mutations seen in cancers are truncations, wherein one or more domains have been deleted. Deletion of the bromodomains might prevent recognition of a specific acetylated‐lysine on a histone, impeding the recruitment of the complete PBAF complex. The mutants without the BAH domains may be unable to ubiquitinate PCNA and so cannot overcome DNA damage. Deletion of the HMG box could prevent the recognition and/or binding of BAF180 to gene sites. It can therefore be inferred that mutations in BAF180, especially truncations, have a key role in the progression of cancer, possibly through the interactions noted above. Here we report the successful expression of the full‐length BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris . The ability to express full‐length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression. Support or Funding Information Michigan Technological University