Expression Vectors and Fusion Proteins
Page 783 sequences in higher eukaryotic genomes. If a subcloned DNA probe contains a repeat sequence it hybridizes to numerous bacteriophage plaques and prevents the identification of a specific overlapping clone. 18.11— Expression Vectors and Fusion Proteins Recombinant DNA methodology described to this point has dealt primarily with screening, amplification, and purification of cloned DNA species. An important goal of recombinant DNA studies, as stated earlier, is to have a foreign gene expressed in bacteria with the product in a biologically active form. Sequencing the DNA of many bacterial genes and their flanking regions has identified the spatial arrangement of regulatory sequences required for expression of genes. A promoter and other regulatory elements upstream of the gene are required to transcribe a gene (Chapter 19, Section 19.3). mRNA transcript of a recombinant eukaryotic gene, however, is not translated in a bacterial system because it lacks the bacterial recognition sequence, the Shine–Dalgarno sequence, required to properly orient it with a functional bacterial ribosome. Vectors that facilitate the functional transcription of DNA inserts, termed expression vectors, have been constructed such that a foreign gene can be inserted into the vector downstream of a regulated promoter but within a bacterial gene, commonly the lacZ gene. The mRNA transcript of the recombinant DNA contains the lacZ Shine–Dalgarno sequence, codons for a portion of the 3 end of the lacZ gene protein, followed by the codons of the complete foreign gene of interest. The protein product is a fusion protein that contains a few Nterminal amino acids of the lacZ gene protein and the complete amino acid sequence of the foreign gene product. Foreign Genes Can Be Expressed in Bacteria Allowing Synthesis of Their Encoded Proteins Many plasmid and bacteriophage vectors have been constructed to permit expression of eukaryotic genes in bacterial cells. Rapidly replicating bacteria can serve as a biological factory to produce large amounts of specific proteins, which have research, clinical, and commercial value. As an example, human protein hormones are produced by recombinant technologies, which serve as replacement or supplemental hormones in patients with aberrant or missing hormone production. Figure 18.20 depicts a generalized plasmid vector for the expression of a mammalian gene. Recall that the inserted foreign gene must be in the form of cDNA from its corresponding mRNA since the bacterial system cannot remove the introns in the premRNA transcript. The DNA must be inserted in register with the codons of the 3 terminal codons of the bacterial protein when creating a fusion protein. That is, insertion must occur after a triplet codon of the bacterial protein and at the beginning of a triplet codon of the eukaryotic gene protein to ensure proper translation. Finally, the foreign gene must be inserted in the proper orientation relative to the promoter to yield a functional transcript. This can be achieved by directional cloning. Eukaryotic proteins synthesized within bacteria are often unstable and are degraded by intracellular proteases. Fusion protein products, however, are usually stable. The fusion protein amino acids encoded by the prokaryotic genome may be cleaved from the purified protein of interest by enzymatic or chemical procedures. An alternative cloning strategy to circumvent the intracellular instability of some proteins is to produce a foreign protein that is secreted. This requires cloning the foreign gene in a vector such that the fusion protein synthesized contains a signal peptide that can be recognized by the bacterial signal peptidase that properly processes the protein for secretion.