Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein

Abstract

Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1), is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn(2+) and not Mg(2+) metal cations for activity.

Figure 1

Cartoon representation of the PI-SceI GFP-fusion protein structure, SDS-PAGE of purified proteins, and illustration of PCR steps performed to create and amplify the PI-SceI-GFP encoding gene. Panel A shows the ribbon structure of GFP and PI-SceI intein bound to its target sequence , . GFP was inserted into the loop containing G117 (the green ball-and-stick model indicated by arrows); this position does not interfere strongly with the intein's functions. Panel B illustrates the use of overlapping primers and the PCR based cloning technique as described in Material and Methods. After the three primary PCR products were combined, the primers VMA-1 and VMA-2 were used to amplify the entire recombinant gene. Panel C, Lane 1: Commassie blue stain of the purified PI-SceI protein, about 51kDa, from E. coli XL1B (DE3) transformed with pET-15b PI-SceI; lane 2: Commassie blue stain of the purified PI-SceI117GFP protein, about 78kDa, from E. coli XL1B (DE3) transformed with pET-28a PI-SceI 117GFP separated on 10% SDS-gel.

Figure 2

Expression and visualization of the GFP inserted into PI-SceI and VMA1 PI-SceI proteins. A and B, E. coli XL1B (DE3) transformed with pET-28a PI-SceI 117GFP vector. C and D, E. coli XL1B (DE3) transformed with pET-28a vma1 PI-SceI 117GFP^3.9kb vector. E and F, E. coli XL1B (DE3) transformed with pET-28a vma1 PI-SceI 117GFP^3kb vector. A, C, and E expression was conducted in LB media at 10^oC for 72 hours while B, D, and F expression was performed in minimal media at 10^oC for 72 hours. Induction and cell grow in minimal media result in expression of soluble protein since the GFP is seen distributed in cytoplasm of the cells. In LB grown cells more expression was detected; however, the expressed proteins, formed inclusion body in parts of cells.

Figure 3

Demonstration of endonuclease activity and immunoblot assay. Panel A: Digestion of the S. cerevisiae V-ATPase catalytic subunit gene (vma1) without intein. The reactions were performed for one hour at 37^oC in PI-SceI reaction buffers in presence of 2.5 mM metal ions. The target site is present in a 1.9kb DNA sequence and after cleavage two fragments of 1.1 kb and 0.8 kb should be observed. Lane 1: Digestion with purified PI-SceI homing endonuclease using the reaction buffer in presence of 2.5mM MgCl₂. Lane 2, 3, 4, and 5: Digestion with purified PI-SceI117GFP using the reaction buffer in presence of 2.5mM MgCl₂, MnCl_2, ZnCl₂, or CaCl₂ respectively. As shown, the GFP-fusion PI-SceI homing endonuclease only cleaves the target site in presence of MnCl₂. Panel B: Immunoblot assay using commercial anti-Sce VMA1 intein antibodies. Lane 1: Purified PI-SceI117GFP protein (positive control). Lane 2: Protein extract from XL1B (DE3) without plasmid (negative control). Lane 3 and 4: Protein extracts from XL1B (DE3) with plasmids expressing PI-SceI 117GFP^3.9kb and PI-SceI 117GFP^3kb respectively. Expression was performed in minimal media as described in the experimental procedure.