Molecular Biotechnology: Principles and Applications - Glick, B., & Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Translational Expression Vectors

Having a strong regulated promoter is a vital, yet insufficient condition for maximizing the yield of a cloned Gene product. Translation efficiency and the Stability of the product itself also play a major role. In Prokaryotic Cells, different mRNAs are not always translated with the same efficiency. This variation can span several hundredfold, resulting in hundreds or even thousands of copies of certain protein molecules within The Cell alongside only a few copies of others.

Differences in translation efficiency are linked—at least in part—to The properties of the Translation initiation signal present in the transcribed RNA, known as the ribosome binding site. A ribosome binding site is a sequence of six to eight NUCLEOTIDES (for instance, UAAGGAGG) that base-pairs with the complementary sequence (in this case, AUUCCUCC) of the RNA component (rRNA) of the small ribosomal subunit. Generally, the stronger the binding between mRNA and rRNA, the higher the translation initiation efficiency. This is why most E. coli expression vectors are engineered so that the mRNA of the cloned gene invariably contains a robust ribosome binding site. This is a prerequisite for expressing heterologous prokaryotic and eukaryotic genes in E. coli. However, several other conditions must also be met. First, The nucleotide sequence that binds to the rRNA must be located at a specific distance from the start codon of the cloned gene (the start codon in RNA is AUG; in DNA, it corresponds to the ATG codon). Second, the DNA segment containing the ribosome binding site and the first few codons of the cloned gene must not form an intrastrand base-pairing Structure post-METABOLISM/31.html">Transcription (Fig. 6.13) that would disrupt mRNA-ribosome binding. In fact, it is the local Introduction/11.html">Secondary structure of the mRNA—which either shields or exposes the ribosome binding site—that determines the binding affinity between the mRNA and the complementary rRNA. Therefore, when cloning any gene, it is crucial to ensure that the ribosome binding site is positioned at the correct distance from the gene and that the mRNA secondary structure does not hinder its attachment to the ribosome.

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Fig. 6.12. Cloning multiple copies of a gene in a single plasmid. A. Construction of the vector. The plasmid is cleaved at the AvaI site, and the resulting sticky ends are filled in using E. coli DNA polymerase I. An EcoRI linker is joined to the blunt ends, recircularizing the plasmid. B. Insertion of the EcoRI linker into the AvaI site of the plasmid. C. Formation of a unidirectional tandem repeat.

Fig. 6.13. Intrastrand base-pairing in an mRNA molecule that hinders efficient translation. GGGGG is the ribosome binding site, AUG (red letters) is the initiation codon, and CAG-CAU-GAU-UUA-UUU represent the first few codons. Note that In addition to the standard A·U and G·C Base Pairs found in mRNA, G-U pairs sometimes form.

Numerous vector systems have already been developed that incorporate both transcriptional and translational signals to ensure the Expression of cloned eukaryotic genes in E. coli. One such system is the pKK233-2 expression vector, which contains the following elements (Fig. 6.14):

✵ an ampicillin resistance Selection marker

✵ a tac promoter

✵ a lacZ ribosome binding site

✵ an ATG start codon located eight nucleotides downstream from the ribosome binding site

✵ phage λ transcription termination sites T1 and T2.

Fig. 6.14. Expression vector based on the pKK233-2 plasmid (not to scale). It contains the ampicillin resistance gene (Amp^r) as a selectable marker, the tac promoter (ptac), the lacZ ribosome binding site (rbs), three restriction endonuclease sites (NcoI, PstI, and HindIII), and two transcription termination sites (T1 and T2). The arrow indicates the direction of transcription.

The cloned gene is inserted into the NcoI, PstI, or HindIII site located between the ribosome binding site and the transcription termination sites. If its reading frame is out of phase with the AUG codon, minor corrections must be made. Following induction and transcription under these conditions, the cloned gene is translated quite efficiently. However, one must bear in mind that because the nucleotide sequence encoding the N-terminal region of the target protein varies among different cloned genes, it is impossible to design a universal vector that completely eliminates single-stranded mRNA base-pairing under all circumstances. Consequently, no translation initiation region, regardless of how optimized it is, can guarantee high translation efficiency for every cloned gene. Thus, the expression vectors described above serve merely as a foundation for developing an optimal translation system.

Efficient translation can also be hindered by "host incompatibility," which arises when the cloned gene contains codons that are rarely used in The Genome of the host Organism. In such cases, the host cell may lack sufficient Transfer RNAs (tRNAs) that recognize these rare codons, thereby diminishing the yield of the cloned gene product. Solving this problem is not always straightforward. If the product of the cloned gene is of high commercial or scientific value, one can attempt to chemically synthesize a variant of the cloned gene composed of codons preferentially utilized by the host organism (codon optimization).