Versatile vectors for direct cloning and ligation-independent cloning of PCR-amplified fragments for surface display on filamentous bacteriophages.
We have constructed phagemid and phage-based vectors which can be used for both direct (T/A) and ligation-independent cloning (LIC) of PCR products for surface display of encoded peptides/proteins fused with the gIII protein of the filamentous bacteriophages M13 and fd-tet. The vectors harbour a DNA cassette consisting of the lacZ alpha fragment inserted between the +2 and +3 codons of gIIIp. The lacZ alpha fragment is flanked by several restriction enzyme recognition sites which can be used for conventional blunt- and cohesive-end cloning in addition to T/A cloning and LIC. The cloning strategies lead to the loss of the lacZ alpha fragment facilitating the selection of the recombinants in XGal plates. The efficiency of direct (T/A) cloning and LIC for surface display in both vectors was evaluated using PCR-amplified fragments encoding a variety of different proteins which included the Fc-binding domain of protein A, the ADP-ribosylation domain of Pseudomonas exotoxin A and a single-chain antibody fragment. The cloning efficiency obtained was 75-85% using the two strategies as monitored by restriction enzyme analysis of the recombinant white colonies on XGal plates. The expression of encoded proteins in recombinants, which were displayed as gIIIp fusions, was found to be 10% in case of T/A cloning but more than 90% in case of LIC.
Sampath
A
A
Department of Biochemistry, University of Delhi South Campus, New Delhi, India.
Abrol
S
S
Chaudhary
V K
VK
D017104:Bacteriophage M13 / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic