Recombinant expression and affinity purification of snake venom gland parvalbumin in Escherichia coli

Parvalbumins (PV) are small, acidic, water soluble and calcium-binding proteins generally present in muscular and nervous tissues. In the present study, we identified and characterized a cDNA clone encoding PV, named ApIPV, from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. ApIPV belongs to EF-hand proteins with six alpha-helices constituting three EF-hand domains. The deduced amino acid sequence of ApIPV is 91% and 68% identical to the previously characterized PVs of Boa constrictor and Cyprinus carpio, respectively. The full-length cDNA was subdoned into the expression vector pGEX and transformed into Escherichia coli (E.coli) to produce recombinant protein. The bacterially expressed GST-ApIPV fusion protein was highly expressed, and effectively purified by Glutathione-Sepharose affinity chromatography. A high concentration of thrombin protease specifically cleaved and removed the GST tag from fusion protein, and further purified by Benzamidine column for removal of thrombin protease. As a result, the 12 kDa ApIPV recombinant protein alone was purified. To investigate the tissue-specific biological occurrence of ApIPV, a polyclonal antibody (anti-ApIPV-antibody) was raised against GST-ApIPV fusion protein in rabbit. Western blot analysis revealed that immunoreactive bands were exhibited in both recombinant protein and samples of venom glands, but not in any crude venom. This specific occurrence indicates a specialized function of ApIPV in snake venom glands. (C) 2009 Elsevier Inc. All rights reserved.