title: A novel method for the purification of low soluble recombinant C-type lectin proteins.
abstract: Snake venoms contain a complex mixture of many biological molecules including proteins. The purification of recombinant proteins is a key step in studying their function and structure with affinity chromatography as the common method used in their purification. In bacterial expression systems, hydrophobic recombinant proteins are usually precipitated into inclusion bodies, and contaminants are typically associated with tagged proteins after purification. The purpose of this study was to develop a procedure to purify hydrophobic recombinant proteins without an affinity tag. Snake venom mature C-type lectin-like proteins (CLPs) with a tag were cloned, expressed, and purified by repeated sonication and wash steps. The effects of the signal peptide on the expression and solubility of the recombinant protein were investigated. The CLPs in washed inclusion bodies were solubilized and refolded by dialysis. The CLPs without a tag were successfully purified with a yield 38 times higher than the traditional method, and inhibited blood platelet aggregation with an IC(50) of 100.57 μM in whole blood. This novel procedure is a rapid, and inexpensive method to purify functional recombinant hydrophobic CLPs from snake venoms useful in the development of drug therapies.
authors.affiliation: Texas A&M University Kingsville, Department of Biological and Health Sciences, Kingsville, TX 78363, USA.
authors.forename: Chunhui
authors.initials: C
authors.lastname: Yin
authors.forename: Ying
authors.initials: Y
authors.lastname: Jia
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Garcia
mesh_terms: D017836:Agkistrodon; D000818:Animals; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry*; D004926:Escherichia coli; D037181:Lectins, C-Type / Q000737:chemistry / Q000302:isolation & purification*; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D012995:Solubility; D013010:Sonication; D014462:Ultrafiltration
chemical_list: D003435:Crotalid Venoms; D037181:Lectins, C-Type; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins