Baculovirus–Insect Cell Expression Systems
Baculovirus Expression Vectors
Mammalian Glycosylation and Processing of Proteins in Insect Cells
Mammalian Cell Expression Systems
Selectable Markers for Mammalian Expression Vectors
Engineering Mammalian Cell Hosts for Enhanced Productivity
Chromosomal Integration and Environment
Site-Directed Mutagenesis by Overlap Extension PCR
Site-Directed Mutagenesis by Inverse PCR
Mutant Proteins with Unusual Amino Acids
Random Insertion/Deletion Mutagenesis
Random Mutagenesis with Degenerate Oligonucleotide Primers
Examples of Protein Engineering
Modifying Cofactor Requirements
Decreasing Protease Sensitivity
FOR MANY BIOTECHNOLOGY APPLICATIONS, a primary objective is to produce high levels of a protein from a cloned gene in a selected host organism. There is no single strategy for obtaining maximal expression of every cloned gene, and many biological parameters must be manipulated to obtain optimal levels of gene expression. These include the genetic elements for controlling transcription, translation, protein stability, and secretion of the product of the cloned gene from the host cell. The level of foreign-gene expression also depends on the host organism. Initially many of the commercially important proteins produced by recombinant DNA technology were synthesized in Escherichia coli. Today many other host systems, such as other bacterial strains, yeasts, and insect and mammalian cells, are employed to produce heterologous proteins. Each of these systems has advantages and disadvantages (Table 3.1). For example, while cloned genes may be expressed at high levels and low cost in E. coli, the proteins produced are not glycosylated. Post translational glycosylation is essential for the function of many human therapeutic proteins, and therefore these proteins are often produced in cultured mammalian cells even though the costs are higher and the yields lower.
Table 3.1 Production of recombinant human proteins in various biological hosts
Protein Production in Prokaryotic Hosts
There are several good reasons to employ prokaryotic cells for production of heterologous proteins, and many bacterial hosts are commercially available such as Gram-negative E. coli and Pseudomonas fluorescens, and Gram-positive Bacillus subtilis, Lactococcus lactis, and Corynebacterium glutamicum. The genetics, molecular biology, biochemistry, and physiology of these bacteria are well understood, they can often be grown to high cell densities in large-scale bioreactors, the growth medium is relatively inexpensive, protocols for their manipulation have been optimized, and vectors are available that carry signals for high levels of gene expression. The latter include the promoter and other transcription regulatory sequences, and sequences that control translation efficiency such as the strength of the ribosome-binding site. Production of a foreign protein in a bacterium may require manipulation of the coding sequence to increase protein stability or direct it to be secreted. To ensure that the cloned gene is maintained in the host cell, it may be necessary to integrate it into the chromosome of the host cell.
Regulation of Transcription
The minimal requirement for an effective gene expression system is the presence of a strong and regulatable promoter sequence upstream from a cloned gene. A strong promoter is one that has high affinity for RNA polymerase, with the consequence that the adjacent downstream region is frequently transcribed (Fig. 3.1). However, a high level of transcription is not always desirable, and the presence of regulatory sequences in the promoter region enables the cell (and the researcher) to control the extent of transcription in a precise manner. Many different promoters with distinctive properties that make them useful for controlling expression have been isolated from a range of organisms (Table 3.2).
Figure 3.1 A strong E. coli promoter resembles the consensus sequences for the −35 and −10 boxes that bind to RNA polymerase. The consensus sequence was determined by aligning many E. coli promoters and identifying conserved nucleotides in the sequences centered −35 and −10 bp upstream of the transcription start site (+1). The distance, but not the nucleotide
