In developing a #biologics product that is inexpensive and efficient, we need to first look at the gene sequence and understand which aspects of it make the product possibly best.
Strategies
Codon optimization: All the different hosts, be it bacteria, yeast, insect or mammalian cell lines, have a set of #tRNAs that are different from each other. So in order to get maximum yield off the host, we need to include codons for amino acids that are more prevalent in the host. Codon optimization now has become pretty easy with lots of free online tools over the internet. #Codon Adaptation Index (#CAI) is the ratio of the usage the codon (wild type/ optimized) has to the frequency of the most frequent codon for the specific amino acid. Values of CAI range from 0 to 1, with higher values indicating higher rate at the most abundant codons. For a codon-optimized sequence, CAI should be close to 1.
Change of gene arrangement: The right order of expression cassette leads to better yield, stability and overall quality of the product. There should be a definite position of #promoters, #enhancers, #modulators, #UTRs and other structural elements in the #expression vector. In case of cloning #mAbs, the constant regions can be taken from a human #cDNA library to boost effectiveness.
Control of proportion of genes: Most #biologics products are #mAbs (monoclonal antibodies) or at large a variant of an antibody which are comprised of a #HC (heavy chain) and a #LC (light chain). Now it needs to be optimized how HC and LC are to be placed in the #expression vector, and also at what ratio.
Change of GC content: Sequence optimization also entails changing the #GCcontent of the whole sequence to approximately 60%. This has been scientifically shown to improve yields in mammalian systems.
Incorporating U depletion: Toning down overall percentage of #Uracil also has been shown to boost efficacy of #biologic products. For example, #Serine has the following codons: UCU, UCC, UCA, UCG, AGU, AGC, but choosing AGC will serve to increase GC content as well as deplete U.
Avoiding restriction enzymes.
Avoiding secondary structure formation.
Hope it helps!
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