You wish to produce a human enzyme, protein A, by introducing its gene into bacteria. The genetically engineered bacteria make l
arge amounts of protein A, but it is in the form of an insoluble aggregate with no enzymatic activity. Which of the following proceduresmight help you to obtain soluble, enzymatically active protein? Explain your reasoning.A. Make the bacteria synthesize protein A in smaller amounts.B. Dissolve the protein aggregate in urea, then dilute the solution and gradually remove the urea.C. Treat the insoluble aggregate with a protease.D. Make the bacteria overproduce chaperone proteins in addition to protein A.E. Heat the protein aggregate to denature all proteins, then cool the mixture.
The reason is that some proteins require molecular chaperones if they are to fold properly within the environment of the cell. In the absence of chaperones, a partially folded polypeptide chain has exposed amino acids that can form non-covalent bonds with other regions of the protein itself and with other proteins, thus causing nonspecific aggregation of proteins.
The option A) is correct because the protein you are expressing in bacteria is being made in large quantities, it is possible that there are not enough chaperone molecules in the bacterium to fold the protein. Expressing the protein at lower levels might increase the amount of properly folded protein.
The option B) is correct as urea should solubilize the protein and completely unfold it. Removing the urea slowly and gradually often allows the protein to refold. Presumably, under less crowded conditions, the protein should be able to refold into its proper conformation.
The option C) is not correct as treating the aggregate with a protease, which cleaves peptide bonds, will probably solubilize the protein by trimming it into pieces that do not interact as strongly with one another; however, chopping up the protein will also destroy its enzymatic activity.
The option D) is correct because overexpressing chaperone proteins might increase the amount of properly folded protein.
The option E) is not correct as heating can lead to the partial denaturation and aggregation of proteins to form a solid gelatinous mass, as when cooking an egg white, and rarely helps solubilize proteins.
Adding lots of organic matter such as compost, farm manure, or shredded leaves to clayey soil will allow it to drain more easily and hold the right amounts of water and air for better plant growth and increased biological activity.
