Direct cloning of polymerase chain reaction products into the pinpoint Xa1-T vector protein expression system

Louise Brown, Jian Lin Yin, Brett Hambly*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)860-866
Number of pages7
JournalElectrophoresis
Volume19
Issue number5
DOIs
Publication statusPublished - May 1998

Keywords

  • Bacterial protein expression vector
  • Biotin affinity chromatography
  • Polymerase chain reaction
  • Protein purification
  • Recombinant proteins

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