Recombinant protein production in bacterial hosts (1)
The bacterial recombinant protein production process is outlined.
Some commonly used promoter systems are described
The pET system, based on the T7 RNA polymerase, is commonly used in recombinant protein production. The pET system relies upon an engineered E. coli host that carries a chromosomal copy of the gene encoding the RNA polymerase of bacteriophage T7. This RNA polymerase gene is usually under the control of an isopropyl b-D-1-thiogalactopyranoside (IPTG) inducible promoter, such as lacUV5.
Maintenance of plasmids is a source of metabolic burden to the host bacterium; therefore, it is usually selected for by the use of an antibiotic and corresponding resistance gene on the plasmid. A general disadvantage of antibiotic selection is the need to eliminate residual antibiotic completely from the generated recombinant protein drug before administration to humans (e.g. to eliminate the risk of allergic reactions).
The balance between high copy number for high recombinant protein productivity and low copy number for decreased metabolic burden must be determined and optimized.
BL21 is a desirable strain because it frequently exhibits a higher biomass yield and lower acetate production compared with K-12. Many complex media contain hydrolysed protein mixtures from animal origin (e.g. tryptone or peptones) and, therefore, are unsuitable for use in the manufacture of human drugs. Yeast- or soybean-derived alternatives are available. Both strategies have found success in different processes; generally speaking, proteins that are known to inhibit growth are synthesised after biomass accumulation, whereas proteins whose synthesis does not cause growth inhibition can be made alongside growth.
Problems can be separated into two broad groups: metabolic problems caused by the process of protein production; and cellular responses to the recombinant protein itself. Commonly referred to as ‘toxic’ recombinant proteins, further investigation reveals several potential solutions, depending upon the root cause of the problem. The problem may not be toxicity caused by the recombinant protein but metabolic problems generated by the transcription and translation process. Each organism preferentially utilizes certain codons; there- fore, a commonly used codon for arginine in humans (AGG) is rarely used in E. coli (for only 2% of arginine codons).
Frequently, if a recombinant protein misfolds, it will form inclusion bodies (IBs), dense particles comprising unfolded and partially folded proteins in varying proportions. Extreme care must be taken when using IB expression and refold pathways that the refolded protein is homogeneously and correctly folded, and folded in the same manner as the native protein.
Affinity tags are very useful for isolating an expressed protein from bacteria but not usually utilized for the production of protein drugs for human use, owing to the requirement to cleave the tag before administration, thus increasing the complexity of downstream processing.
The Crohn’s disease and rheumatoid arthritis treatment Cimzia1 (UCB) is an anti-tumour necrosis factor (TNF)-a Fab’ fragment made in E. coli and conjugated to PEG.
