Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.
The use of the synthetic drugs has increased in the last few decades; however, these drugs exhibit various side effects. Scientists are therefore seeking alternatives from natural sources. Commiphora gileadensis has long been used to treat various disorders. It is commonly known as bisham or balm of Makkah. This plant contains various phytochemicals, including polyphenols and flavonoids, with biological potential. We found that steam-distilled essential oil of C. gileadensis exhibited higher antioxidant activity (IC50, 22.2 µg/mL) than ascorbic acid (IC50, 1.25 µg/mL). The major constituents (>2%) in the essential oil were β-myrcene, nonane, verticiol, β-phellandrene, β-cadinene, terpinen-4-ol, β-eudesmol, α-pinene, cis-β-copaene and verticillol, which might be responsible for the antioxidant and antimicrobial activity against Gram-positive bacteria. The extract of C. gileadensis exhibited inhibitory activity against cyclooxygenase (IC50, 450.1 µg/mL), xanthine oxidase (251.2 µg/mL) and protein denaturation (110.5 µg/mL) compared to standard treatments, making it a viable treatment from a natural plant source. LC-MS analysis revealed the presence of phenolic compounds such as caffeic acid phenyl ester, hesperetin, hesperidin, chrysin and transient amounts of catechin, gallic acid, rutin and caffeic acid. The chemical constituents of this plant can be explored further to investigate its wide variety of therapeutic potential.
Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3–5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.
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