Question

A bacterium isolated from the deep ocean floor was found to produce a protein that was soluble in aqueous solutions, and could effectively breakup plant material to produce ethanol, a biofuel, when present as a homodimer. However, the protein rarely forms dimers. Assuming the gene for this protein has been cloned, and using the material discussed in BIOL 366, propose a mechanism to engineer and produce the recombinant form of this protein such that (i) it readily forms homodimers, and (ii) can be purified by affinity chromatography.

Answer 1

Loss of function analyses, misexpression and dosage experiments, and biochemical studies indicate that heterodimers of Twist and Daughterless repress genes required for somatic myogenesis. We propose that these two opposing roles explain how modulated Twist levels promote the allocation of cells to the somatic muscle fate during the subdivision of the mesoderm. Moreover, this work provides a paradigm for understanding how the same protein controls a sequence of events within a single lineage.

Several lines of experimentation suggest that Twist forms dimers that have distinct functions during development. Genetic studies have revealed two twist alleles (twistv50 and twistry50) that are temperature sensitive only when in trans with one another. Neither allele is temperature sensitive by itself or over a deficiency, but twistv50/twistry50 survive at 18°C and die at 29°C. This suggests that the two mutant proteins form a temperature sensitive dimer. Temperature shift experiments indicate that this homodimer functions early, to direct specification of mesoderm , and during allocation of somatic muscle . These results also suggest that Twist homodimers are capable of activating mesodermal and somatic muscle specific genes.

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There are numerous ways in which an affinity-based method may be employed for the purification of recombinant proteins. The most common example of an affinity process is protein-A chromatography, which has been applied for over a decade in industrial and academic settings for the capture and purification of antibodies (immunoglobulins) . In spite of several notable drawbacks associated with the use of this technology, primarily the ability of protein-A to leak into the mobile phase and the high associated costs, it is still widely used as a capture step in large-scale purification of monoclonal antibodies (mAbs), including those of therapeutic use . Recombinant protein-A-bound resins with high binding capacities are commercially available and can withstand the harsh sterilising conditions introduced between cycles of industrial downstream processing. What makes protein-A-based applications more lucrative for industrial uses, in spite of their obvious disadvantages, is their ability to indirectly remove viruses from the feed . Moreover, the ability to capture mAbs directly from clarified harvest without any pretreatment and very high selectivity leading to removal of most host cell proteins are two distinct advantages of protein-A chromatography.