title: Direct cloning of polymerase chain reaction products into the pinpoint Xa1-T vector protein expression system.
abstract: Expression of recombinant proteins is an important method for the characterisation of the structure and function of proteins. However, many expression methods can be difficult, time-consuming and lead to low protein yields. The Promega Pinpoint Xal-T vector system is a unique, one-step cloning method that allows the direct insertion of polymerase chain reaction (PCR) fragments into the expression vector. We describe our experience of the use of this system to clone and express three proteins (8-12 kDa) directly from their PCR products. The proteins are expressed as fusion proteins with a 13 kDa biotinylated tag that can be used for detection of the expressed protein and affinity purification. In our case, the yield was greater than 20 mg per litre of culture. Expressed proteins were purified by Q-Sepharose anion-exchange chromatography and reverse-phase high-performance liquid chromatography (HPLC) instead of the conventional method of avidin-biotin affinity chromatography. The Pinpoint vector proved to be a relatively simple and fast protein expression technique suitable for wide application for expressing recombinant proteins.
authors.affiliation: Department of Pathology, University of Sydney, Australia.
authors.forename: L
authors.initials: L
authors.lastname: Brown
authors.forename: J L
authors.initials: JL
authors.lastname: Yin
authors.forename: B
authors.initials: B
authors.lastname: Hambly
mesh_terms: D000818:Animals; D001710:Biotin; D002352:Carrier Proteins / Q000235:genetics; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D000099163:Myosin Binding Protein C; D000099255:Growth Arrest-Specific Protein 6
chemical_list: D001710:Biotin; D002352:Carrier Proteins; D036341:Intercellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000099163:Myosin Binding Protein C; D000099255:Growth Arrest-Specific Protein 6