Cloning, expression, crystallization and preliminary X-ray studies of a superfolder GFP fusion of cyanobacterial Psb32

Abstract

A fusion of Psb32 from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePsb32) with superfolder GFP was created for enhanced solubility and improved detection and purification. The fusion protein readily formed large hexagonal crystals belonging to space group P6₁22. A full data set extending to 2.3 Å resolution was collected at the Swiss Light Source. The phase problem could be solved by using only the sfGFP fusion partner or by using GFP and AtTLP18.3 from Arabidopsis thaliana as search models. Based on this expression construct, a versatile library of 24 vectors combining four different superfolder GFP variants and three affinity tags was generated to facilitate expression and screening of fluorescent fusion proteins.

Keywords: Psb32; Thermosynechococcus elongatus BP-1; superfolder GFP.

Figure 1

Schematic representation of the modular architecture of the presented plasmids. Plasmid variants were engineered for the expression of the protein of interest (POI) with different affinity tags (6×His-tag, Strep-tag II or Twin-Strep-tag) and superfolder GFP colour variants at (a) the amino-terminus or (b) the carboxy-terminus. Plasmid pStrepGFP-TePsb32 used for expression of STII-sfGFP-TePsb32 (with TePsb32 being the POI) is highlighted by a red dashed line. The ‘X’ in the plasmid name serves as an indicator of the respective colour variant: G, green; Y, yellow; B, blue; C, cyan.

Figure 2

Native STII-sfGFP-TePsb32 appeared as a single protein of around 48 kDa on 15% SDS–PAGE after elution from a RESOURCE Q column. Fermentas PageRuler Prestained Protein Ladder was used as a marker.

Figure 3

Hexagonal rod-shaped crystal of STII-sfGFP-TePsb32 (400 × 50 × 45 µm). Crystallization was performed by hanging-drop vapour diffusion in 0.75 M sodium citrate, 0.1 M CHES pH 9.5 at 291 K using a protein concentration of 25 mg ml⁻¹.

Figure 4

Analysis of dissolved crystals of STII-sfGFP-TePsb32 on 15% SDS–PAGE demonstrates the integrity of the fusion protein. Proteins were stained with Coomassie Brilliant Blue or irradiated with UV light. Lane 1 contains the protein solution, lane 2 contains Fermentas Unstained Protein Molecular Weight Marker and lane 3 contains redissolved protein crystals.