Cloning, Purification, and Partial Characterization of the Halobacterium sp. NRC-1 Minichromosome Maintenance (MCM) Helicase

Abstract

The MCM gene from the archaeon Halobacterium, with and without its intein, was cloned into an Escherichia coli expression vector, overexpressed and the protein was purified and antibodies were generated. The antibodies were used to demonstrate that in vivo only the processed enzyme, without the intein, could be detected.

Fig. (1)

Alignment of full length Halobacterium MCM protein. Amino acid sequence alignment of five archaeal MCM helicases: Halobacterium sp. NRC-1, S. solfataricus, T. acidophilum, M. thermautotrophicus, and A. fulgidus. Highlighted residues are conserved in all five proteins. The long inserted sequence found in Halobacterium sp. NRC-1 is the intein.

Fig. (2)

Purification of the Halobacterium MCM helicase. Uninduced and induced E. coli cells harboring pET-MCM (lanes 2 and 3) and pET-MCMint (lanes 5 and 6) and the purified MCM protein (0.5 µg) with intein (lane 4) and without intein (lane 7) were fractionated on 10% SDS-PAGE. Lane 1, molecular weight markers.

Fig. (3)

Mass spectroscopy analysis of the E. coli expressed proteins. MALDI-TOF spectra of Halobacterium MCM with (A) and without (C) intein were performed as described in “Material and Methods”. The 2158.16 and 2333.16 peaks were analyzed using MS/MS (C and D,respectively) as described in “Material and Methods”. Panel E shows the position of the peptide in the MCM amino acid sequence. Shaded areas show the location of the intein. The peptide corresponding to the 2158.16 peak is underlined and that for the 2333.16 peak is double underlined.

Fig. (4)

Only MCM protein without intein can be detected in Halobacterium cells. Western analysis of Halobacterium cell extract and recombinant MCM protein was performed as described in“Material and Methods”. Lane 1, cell extract (1 µg); lane 2, cell extract (0.5 µg); lane 3, purified MCM protein without intein (1ng); lane 4, purified full length MCM (20 ng). To the left are the positions of the molecular weight markers.