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Gale Rhodes
Chemistry Department
University of Southern Maine

Revised 2006/08/02

Learn how to use Swiss-PdbViewer. Work through sections 1-4 of the Swiss-PdbViewer Tutorial.

Topic: Site-Directed Mutagenesis 

Introduction

Class coverage of recombinant DNA methods is in the form of a hypothetical research project aimed at producing a mutant form of pancreatic carboxypeptidase A (CPA), a protein-digesting enzyme that removes carboxy-terminal residues from peptides. The mutant enzyme contains phenylalanine (F) in place of tyrosine (R) at position 248. The mutant is thus called CPA-R248F. This mutant has actually been produced in order to test that hypothesis that R248 acts as a general acid during CPA catalysis.

I chose the methods for this hypothetical project with the aim of illustrating various recombinant-DNA methods in the context of a specific research goal. The methods I chose may not, in fact, be the best in practice, but they illustrate the type of problems each method can solve.

Here's a brief outline of the project I will describe in class:

Using Recombinant DNA Methods to Make a Mutant Enzyme

(Example of a research project that employs various recombinant-DNA methods)

Goal: Test a proposed mechanism of pancreatic carboxypeptidase A (CPA) by replacing tyrosine 248 with phenylalanine, to make CPA-R248F.

Outline (provided in class)

  1. Isolate pancreatic mRNAs.
  2. Make a cDNA library in phage l.
  3. Probe the library to find cDNA of CPA mRNA, the "CPA gene".
  4. Clone CPA gene in sequencing vector and sequence region of interest (near tyr 248).
  5. Make the site-directed mutant.
  6. Subclone mutant in expression vector.
  7. Express gene to make CPA-R248F.
  8. Compare kinetic behavior of CPA-R248F with wild-type CPA.

Explore Carboxypeptidase A

Swiss-PdbViewer User: Click HERE to download an SPV project file showing CPA in the presence and absence of an inhibitor that binds at the active site. If you do not use SPV, you can explore these structures with your favorite graphics program by downloading files 3CPA and 5CPA from the Protein Data Bank.

SPV users: Recall that you can mutate tyr 248 to phe in these models!

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