Abstract
Marker-free gene replacement is a useful technique for modification of chromosomal genes without limitation of the availability of drug-resistant markers. By using this method, we introduced an affinity tag for easy purification of photosystem II (PSII) in cyanobacterial strain Synechococcus elongatus PCC 7942.
In order to add histidine tag (histag) at the C-terminus of CP47 subunit of PSII, we transformed a streptomycin-resistant strain GRPS1 (rps12-R43) from S. elongatus PCC 7942 R2-SPc in two-step protocol. Because psbB gene encoding CP47 is involved in psbB-psbT operon, we constructed not only GRPS800 strain carrying an insertion of [rps12-kan] cassette at the 3' end of psbB gene but also GRPS801 strain carrying psbB promoter upstream the psbT gene. In the second transformation, [rps12-kan] cassette was removed, giving rise to a marker-free GRPS810 strain. Analysis of solubilized proteins from thylakoid membrane from GRPS810 by metal-chelate affinity chromatography confirmed that this strain is useful for one-step purification of PSII complex with CP47-histag modification.
