Cloning, expression and purification of Y87G2A.14
The C. elegans Y87G2A.14 gene encodes a 234 amino acid protein with an expected molecular weight of 26,601 Da. It was amplified by PCR from a C. elegans cDNA library. The PCR fragment was inserted into the pET-32b(+) expression vector and the nucleotide sequence of the insert was determined to be exactly the same as that submitted to GenBank under accession no. CAB54476. The recombinant plasmid pETY87G2A.14 was then used to transform E. coli BL21 (DE3) cells to generate a His-tagged thioredoxin fusion protein with an expected molecular mass of 43,731 Da. When the Trx-Y87G2A.14 fusion protein was expressed at 37°C, it was confined to inclusion bodies, so the induction temperature was decreased to 25°C to enhance protein solubility. As the expression level was low at this temperature, the induction time was increased to 8 h. These conditions markedly increased the solubility of Trx-Y87G2A.14 which was then purified from the soluble fraction (Fig 1, lane 2) to apparent homogeneity on NiCAM™-HC resin (Fig 1, lane 3). To determine the molecular weight of the Y87G2A.14 protein itself, the Trx-Y87G2A.14 fusion was cleaved with thrombin, which generated Y87G2A.14 with an apparent molecular weight of 27 kDa (expected molecular weight, 29,807 Da) and thioredoxin (15 kDa, Fig 1, lane 4).
Figure 1  Purification and cleavage of Trx-Y87G2A.14 fusion protein. Samples were analysed by SDS-PAGE (15% polyacrylamide) and stained with Coomassie Brilliant blue R 250. Lane 1, 2 μg protein standards: bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa) and α-lactalbumin (14.2 kDa); lane 2, soluble cell extract of BL21 (DE3) cells transformed with recombinant plasmid pETY87G2A.14 and induced with 1 mM IPTG for 8 hours at 25°C before applying to a column of NiCAM™-HC resin ; lane 3, 3 μg purified Trx-Y87G2A.14 fusion protein; lane 4, 3 μg purified Trx-Y87G2A.14 fusion protein after cleavage with thrombin.