1) A single-chain enzyme of molecular weight 25 kDa has been chosen for use in an industrial process which will take place at a temperature above the melting temperature, of the enzyme. To increase the operational half-life of the enzyme at the process temperature, it has been decided to attempt to increase its stability by introducing a disulfide bridge. The enzyme has been cloned but its three-dimensional structure is not available. However, both the primary and high resolution crystal structures are available for a highly homologous enzyme from the same family.

A. Explain how the primary and tertiary structural information available can be used to generate a model for the structure of the target enzyme.

B. Describe how you might use this model to help decide which residue(s) to mutate so as to maximize the chance of introducing a disulfide bridge into the enzyme.

C. An expression vector has been prepared incorporating the gene for the putative disulfide-containing mutant enzyme. The mutant enzyme will be expressed in frame as a fusion with a C-terminal histidine affinity tag. You now wish to produce milligram-scale quantities of the mutant enzyme. Following its over-expression in E.coli, a cell free extract containing the mutant has been prepared. The fusion protein has a pI of 8.0 and has a molecular weight of 50 kDa. Design a purification strategy for the enzyme explaining the considerations you have taken into account in doing so. The target purity of the purified protein is at least 99.9 % (w/w). Your purification strategy should:

i. take into account how you would determine purity of the final product, ensure that you have obtained the protein of interest, and how you would address issues of contamination since the target purity is at east 99.9 %.

ii. describe the equipment a well-equipped laboratory would require to isolate and purify the protein.

d. Describe briefly how you might determine the change in melting temperature for the purified mutant relative to that of the wild type enzyme. Provide at least TWO experimental methodologies you could use.

e. Introducing a disulfide bridge is one means by which protein stability may be enhanced. Provide rationalizations for TWO other approaches which may be employed.
 

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A Computeraided protein design CAD and homology modeling are two methods that can be used to generate a model for the structure of the target enzyme based on the primary and tertiary structural inform... View the full answer