The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning.
In this chapter, protocols for the construction of expression vectors using In-Fusion PCR cloning are presented. The method enables vector and insert DNA sequences to be seamlessly joined in a ligation-independent reaction. This property of the In-Fusion process has been exploited in the design of a suite of multi-host compatible vectors for the expression of proteins with precisely engineered His-tags. Vector preparation, PCR amplification of the sequence to be cloned and the procedure for inserting the PCR product into the vector by In-Fusion are described.
Berrow
Nick S
NS
Oxford Protein Production Facility, Welcome Trust Centre for Human Genetics, Oxford, UK.
Alderton
David
D
Owens
Raymond J
RJ
D000818:Animals; D018929:Cell Culture Techniques; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D005990:Glycerol / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D006639:Histidine; D005990:Glycerol