Cloning expression vector construction

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

Yen, K. M. (1991). Construction of cloning cartridges for development of expression vectors in gram-negative bacteria. Journal of Bacteriology, 173, 5328-35. 

Nakamura, K., Iwasaki, Y. Hattori, T. (1980). An improved Escherichia coli expression vector for the construction and identification of full-length cDNA clones. Gene 44, 347-51. 

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