This project was focused on characterization of a cysteine-rich motif (C-domain) in the human L1 reverse transcriptase (L1 RT). The L1 RT is a large multifunctional polymerase which plays a key role in driving reverse transcription and integration of the human mobile L1 element. This 150 kDa enzyme consists of an N-terminal domain of AP-like endonuclease, a reverse transcriptase domain and a C-terminal domain of unknown function containing a putative zinc binding motif.
To determine the role of the C-domain the structure needs to be characterized. Performing limited proteolysis, MALDI-TOF-MS, peptide sequencing and alignment and three-dimensional structural modeling could help understanding the function of the C-domain. However, due to the protein nature it is very challenging to produce and purify. The C-terminus domain is a basic protein with strong hydrophobicity. We had to examine the expression level for different recombinants of the C-domain fused with leader sequences of different length to obtain satisfying yields of the target protein in bacteria. The two step purification protocol has been developed de novo. We have tested different pH conditions and salt concentrations for gel filtration, ion-exchange and affinity chromatography. The purified fractions were analysed by SDS-HEPES-PAGE followed by Western Blot.
Further identification of the C-domain, among the bands with similar molecular weight has been performed with MALDI-TOF MS. The C-domain has been purified to near 90% purity.
To evaluate the potency of the L1 ORF2p as a marker for cancer cells, a theoretical protein digest of the C-domain has been performed with Swiss-Prot Peptide Cutter tool followed by BLAST search. A unique signature peptide has been identified by comparison theoretical digest with the experimental MS data.