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Direct cloning of polymerase chain reaction products into the pinpoint Xa1‐T vector protein expression system
Author(s) -
Brown Louise,
Yin Jian Lin,
Hambly Brett
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150190543
Subject(s) - biotinylation , fusion protein , recombinant dna , affinity chromatography , expression vector , cloning (programming) , avidin , microbiology and biotechnology , flag tag , target protein , polymerase chain reaction , chemistry , biology , cloning vector , biochemistry , chromatography , vector (molecular biology) , gene , enzyme , computer science , programming language
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 Xa1‐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.