LGN, named for the many repeats of the amino acids leucine (L), glycine (G) and asparagine (N), is crucial for mammalian cellular division. Specifically, LGN plays a significant role in cell polarity and alignment of the mitotic spindles and in its absence, the organism ceases to develop. In breast cancer, LGN is upregulated and the knockdown of LGN activity has been shown to suppress growth of breast cancer cells. Furthermore, the mutation T450A suppressed breast cancer cell growth.  The goal of this project was to better understand how phosphorylation of T450 affects cell division by protein crystallization, in which the initial step was to overexpress and attain 95% purity before crystals can be grown. To date, full-length LGN has only been isolated in the presence of Gαi and the end product was not 95% pure. Furthermore, several crystal structures of LGN have been solved, but none are of the full-length LGN or contain the T450 residue. We discovered that overexpression of LGN in <i>E. coli </i>cells resulted in significant degradation of the protein, so we moved to a mammalian system for expression. Wild-type LGN has been successfully expressed transiently in baby hamster kidney (BHK)-570 tissue culture cells using a pCMV-LGN plasmid and LipoD 293 reagent.  LGN was isolated by performing a modified immunoprecipitation in which a rabbit anti-LGN polyclonal antibody and Protein A/G Plus-Agarose beads captured the LGN protein and a synthetic LGN peptide released the LGN protein. We estimated that 100 ng of LGN protein was released using the current protocol, so we are currently optimizing it to obtain minimally 0.25 mg of LGN protein after scaling up the process. Furthermore, the 75 kDa LGN protein was less than 50% pure, so subsequent purification methods are being utilized, including gel filtration. The T450A and T450D mutants, which mimic unphosphorylated and phosphorylated LGN, were generated by PCR and verified for sequencing. Ultimately, solving the crystal structures of wild-type and mutant LGN will facilitate the process of novel treatments for breast cancer.

LGN protein purification for X-ray crystallography

253rd National Meeting (2017)

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