Difference between revisions of "Part:BBa K243032"

Revision as of 00:56, 22 October 2009

Fok_a-Venus

This part is like the part protein domain Fok_a from our universal endonuclease, but it has an additional linked YFP-marker.

Usage and Biology

The addition of a fluorescent protein(Venus) to the protein construct enables the detection by fluorescence microscope and a new way of purification by GFP-trap column. The Venus protein was fused C'-terminal to the protein domain, so that when the protein is expressed the fluorescence signal of the Venus protein can be seen.

Detection

There was a strong fluorescence signal under the fluorescence microscope after induction of the cells with IPTG. The E.colis(RV308) were excited with light of the wavelength 505nm and then some pictures of the fluorescence were made.

The cell extracts were checked by SDS-page to get information about the expression of proteins.

SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced)

In the lanes of the induced clones ( lanes 3,4) a band of about 50 kDA appears which could be the Venus-Fok_a construct.

A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP)was conducted to prove the complete expression of the protein+YFP construct and to exclude a degradation of the construct.

Western Blot; pEx Venus-Fok_a in BL21de3; lanes: Marker (NEB prestained protein marker broad range, clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-DigA-SplitLi-Fok_a) uninduced, control induced)

All of the induced clones show a strong signal but also many different bands on the membrane. There is a signal at 49 kDa which is the size of the Venus-Fok_a complex. The bands indicating proteins of smaller size could be degraded proteins.

Purification

It is possible to use the GFP tag or as in our case YFP for purification by a GFP specific column.
Due to the lack of time at the end, the purification of the Fok_a proteins by a GFP column is under progress.

Sequence and Features