ISSN:
0173-0835
Keywords:
Bacterial protein expression vector
;
Recombinant proteins
;
Polymerase chain reaction
;
Protein purification
;
Biotin affinity chromatography
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150190543
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