Optimizing Expression of Recombinant Genes

OPTIMIZING EXPRESSION OF RECOMBINANT GENES

The primary objective of pharmaceutical companies involved in the production of recombinant drugs is the maximal expression of recombinant genes to generate large quantities of these drugs. Unfortunately, the cloning of a gene into a vector does not ensure that it will be properly expressed. To improve expression of a cloned gene the different stages that lead to the synthesis of the protein therefore have to be optimized. This is achieved by the use of so-called expression vectors (Figure 25.6). Some expression vectors have been designed to produce large quantities of protein in specific cell hosts. For example, the bacmids are shuttle expression vectors derived from a baculovirus ds DNA circular genome and are used to transfect insect cells in order to produce large quantities of a recombinant protein in fermenters.

A)   Optimizing Transcription

To optimize transcription it must be ensured that the recombinant gene is placed after a promoter (Figure 25.6) that will be recognized by the RNA polymerase of the host cell where the gene is going to be expressed. Two types of promoters can be used: (1) constitutive promoters, which are expressed all the time and (2) inducible promoters, where expression is turned off during culture growth and turned on, for example, upon the addition of an inducing molecule to the culture, usually shortly before harvesting, when high numbers of bacteria are present in the culture. Inducible promoters are very useful when expressing foreign genes coding for proteins toxic to the bacterial hosts as their premature expression could lead to growth impairments and consequently low yields of recombinant protein.

Furthermore, to ensure that transcription finishes after the 3′ end of the recombinant gene, a transcriptional terminator (Figure 25.6) must be placed just downstream of this gene.

B)   Optimizing Translation

A key feature that determines whether a gene is going to be efficiently translated by a certain host is the nucleotide sequence of the ribosome binding site (RBS), located upstream of the gene (Figure 25.6), which needs to be efficiently recognized by the ribosomes of this host. In addition, the distance between the RBS and the translation start codon needs to be optimal to enable the right interactions between the mRNA and the ribosomes and start the protein synthesis. There are commercially available vectors carrying sequences for RBSs and translation start codons which are optimally recognized by the ribosomes of the host cells, ensuring that any recombinant genes cloned after the start codon will be maximally translated.

Small proteins are frequently susceptible to proteolytic degradation when expressed in a foreign host. This degradation can be avoided by expressing them fused to a larger protein. This is normally achieved by cloning the small gene downstream of a gene coding for a protein such as β-galactosidase. To  obtain the fusion protein (Figure 25.6) it is essential to ensure that the reading frame is conserved and that no translation stop codons are present between the β-galactosidase and the target gene, enabling the ribosomes to read through. Interestingly, affinity columns that will bind the fused polypeptides are available, which facilitates the purification of the recombinant protein by affinity chromatography.

C)    Post-Translational Modifications

Although high levels of protein production may be achieved by optimizing transcription and translation of a gene, the obtained protein may still need to undergo post-translational modifications before it is active. Some of these modifications include correct disulphide bond formation, proteolytic cleavage of a precursor, glycosylation and additions to amino acids such as phosphorylation, acetylation, sulphation, acylation, etc. Unfortunately, the practical E. coli host, in which most recombinant proteins are produced, does not possess the same type of cellular machinery required for these modifications. Recently, Campylobacter jejuni has been found to possess an eukaryotic-like system for protein glycosylation and efforts are being made to genetically engineer E. coli strains to perform the adequate glycosylation of recombinant proteins as mammalian cells. Hence, it is essential to select a suitable host for the expression of the target gene that can carry out the required post-translational modifications that will enable the synthesis of large amounts of a biologically authentic product. Table 25.3 shows a comparison of a selection of hosts currently used for the expression of recombinant proteins.