S. pombe expression vector with 6x(His) tag for protein purification and potential for ligation-independent cloning.

In the area of protein production and purification, researchers have turned to eukaryotic organisms, such as Schizosaccharomyces pombe, as hosts for expression of many eukaryotic proteins instead of bacterial organisms. The reasons are many: some bacterial hosts cannot manage eukaryotic genes due to their toxicity, insolubility or their need for posttranslational modifications, such as phosphorylation and acetylation. It is in this area that S. pombe is greatly utilized due to its unique traits that closely resemble higher eukaryotic organisms. This includes chromosomal structure and function, cell-cycle control and RNA splicing (15). We have constructed and tested a new plasmid vector, pESP-5 (Figure 1), for gene expression and protein production in S. pombe. pESP-5 is derived from pESP-2 (9). This vector contains the S. pombe inducible nmt1 promoter for high-level gene expression (3,11,12) and the 6×(His) affinity tag for protein purification. Similar vectors that contain the 6×(HIS) and GST purification tags and the c-myc, HA and Pk epitope tags have also been constructed for use in fission yeast (2,4). A novel aspect of pESP5 includes the potential of ligation-independent cloning (LIC) (1). The gene of interest can be cloned in the multiple cloning sites by using the conventional restriction fragment ligation method or the LIC method (1,5,7). The LIC strategy allows efficient and directional cloning of the target gene. When specific polymerase chain reaction (PCR) primers are used, the target gene can be placed immediately downstream of the DNA sequences encoding for the epitope tag, FLAG (Scientific Imaging Systems [Eastman Kodak], New Haven, CT, USA) (6), and sequences for recognition by enterokinase. Because enterokinase cleaves the peptide bond C terminal to its recognition site (8), polypeptides with native amino acid sequences can be obtained after removal of the 6x(His) tag by enterokinase. To prepare pESP-5 for LIC cloning, the vector was linearized with SphI and treated with Pfu DNA Polymerase (Stratagene, La Jolla, CA, USA) in the presence of 1 mM dTTP. Pfu DNA polymerase has both 5′ to 3′ polymerase activity and 3′ to 5′ exonuclease activity (10). The 3′ to 5′ exonuclease activity of Pfu DNA polymerase removes nucleotides at the 3′ ends of the vector until a dTMP residue is reached, resulting in a vector with 5′ extended tails of defined sequence and length at both ends. Because the two tails are noncomplementary to each other, the “LIC-ready” vector ensures directional cloning of the insert with low background, avoiding self-ligation of the vector. The chicken calmodulin gene was used as a test gene for cloning into pESP-5 using the LIC cloning strategy. Two specifically designed primers with defined “LIC-specific” tails were synthesized and used to PCR-amplify the chicken calmodulin gene. After 25 cycles of amplification with Pfu DNA polymerase in a RoboCycler 40 (Stratagene), the PCR mixture was electrophoresed on a 1% agarose gel, the gel slice containing the PCR product was excised, and the DNA was recovered using a StrataPrep Gel Extraction Kit (Stratagene). The purified PCR fragment was further treated with Pfu DNA polymerase in the presence of dATP to generate the LIC-specific tails. Pfu DNA polymerase was subsequently removed by extraction with StrataClean Resin (Stratagene). The LIC-ready pESP-5 vector and the LIC-ready insert DNA fragments can then be annealed at room temperature and directly used for transformation. The length of complementary sequences between the vector and the insert (12 and 13 bases, respectively) are long and stable enough to eliminate the ligation step. The annealed mixture was transformed into Escherichia coli cells and selected on LB+amp plates. The cloning efficiency of the calmodulin gene into pESP-5 using LIC strategy was determined by E. coli colony PCR, with 92% containing an insertion at the size expected of the calmodulin gene (data not shown). pESP-5-calmodulin plasmid DNA was then transformed into the S. pombe strain SP-Q01. One microgram of the expression construct was mixed with 100 μL of cells (ca. 2 × 108 cells) of the Benchmarks