METHODS AND RESULTS: This review discusses optimizations and techniques for antibody production through choice of discovery platforms, expression systems, cell culture mediums, and other strategies to increase expression yield. Each system has its own merits and demerits, and the strategy chosen is critical in addressing various biological aspects.
CONCLUSIONS: There is still insufficient evidence to validate the efficacy of some of these techniques, and further research is needed to consolidate these industrial production systems. There is no doubt that more strategies, systems, and pipelines will contribute to enhance biopharmaceutical production.
METHODS: For identification of the minimal selective domain for apoptosis, the wild-type Apoptin gene had been reconstructed by PCR to generate segmental deletions at the N' terminal and linked with nuclear localization sites (NLS1 and NLS2). All the constructs were fused with maltose-binding protein gene and individually expressed by in vitro Rapid Translation System. Standardized dose of proteins were delivered into human breast adenocarcinoma MCF-7 cells and control human liver Chang cells by cytoplasmic microinjection, and subsequently observed for selective apoptosis effect.
RESULTS: Three of the truncated Apoptin proteins with N-terminal deletions spanning amino acid 32-83 retained the cancer selective nature of wild-type Apoptin. The proteins were successfully translocated to the nucleus of MCF-7 cells initiating apoptosis, whereas non-toxic cytoplasmic retention was observed in normal Chang cells. Whilst these truncated proteins retained the tumour-specific death effector ability, the specificity for MCF-7 cells was lost in two other truncated proteins that harbor deletions at amino acid 1-31. The detection of apoptosing normal Chang cells and MCF-7 cells upon cytoplasmic microinjection of these proteins implicated a loss in Apoptin's signature targeting activity.
CONCLUSIONS: Therefore, the critical stretch spanning amino acid 1-31 at the upstream of a known hydrophobic leucine-rich stretch (LRS) was strongly suggested as one of the prerequisite region in Apoptin for cancer targeting. Identification of this selective domain provides a platform for developing small targets to facilitating carrier-mediated-transport across cellular membrane, simultaneously promoting protein delivery for selective and effective breast cancer therapy.
RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.
CONCLUSION: The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii.