Expression vectors for enzyme restriction- and ligation-independent cloning for producing recombinant His-fusion proteins.
In this work we have constructed two novel expression vectors, designated as pURI2 and pURI3, which enable parallel cloning of a given target gene for producing recombinant His-fusion proteins. The vectors were created using the well-known pT7-7 and pIN-III-A3 plasmids as their template. The same DNA fragment containing the His-tag, enterokinase cleavage site, and a NotI unique site, as well as keeping the HindIII unique restriction site, was introduced in both vectors. These vectors have been designed to avoid the enzyme restriction and ligation steps during the cloning. The unique NotI site was introduced to facilitate the selection of the adequate recombinant plasmid. Parallel cloning of the same polymerase chain reaction fragment can be carried out since both vectors shared the same leader sequence. The described strategy avoids tedious cloning efforts into different expression vectors and represents a highly efficient means of cloning. To validate our vectors, we have cloned one target gene in both vectors and used expression and purification techniques to obtain the recombinant target protein. We herein show that both vectors function effectively in all the required experimental steps-cloning, expression, purification, and cleavage.
de las Rivas
Blanca
B
Departamento de Microbiología, Instituto de Fermentaciones Industriales, and Grupo de Cristalografía Macromolecular y Biología Estructural, CSIC, 28006 Madrid, Spain.
Curiel
José Antonio
JA
Mancheño
José Miguel
JM
Muñoz
Rosario
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006639:Histidine / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006639:Histidine; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases; D001616:beta-Galactosidase