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4. Protein synthesis and protein engineering Page 15
If we are to make long polypeptide chains with a-acid monomers in a specified sequence then we must have a very tight control of the reactions we use. In this chapter you will see the complex chemical systems that organisms use and how scientists use the techniques of protein engineering

to tailor modifications. But first we will look at the more conventional, though elegant, approach of the synthetic organic chemist.

4.1 Solid phase peptide synthesis

The synthetic organic chemist faces several problems:

1. The carboxylic acid group (-COOH) in an a-amino acid is not reactive enough to give a condensation reaction with the amine group (- NH2) of another amino acid using mild conditions.
2. As the polypeptide chain is built up unit by unit, care must be taken not to expose it to conditions that would hydrolyse the peptide links already made.
3. Many a-acids have side chains with functional groups that may react under the conditions used to make the peptide links.

diagram of adding units to correct end
4. A polypeptide chain has two reactive ends; the free -NH2 and the free -COOH. The synthetic chemist must make sure that each extra a -amino acid unit adds on to the correct end of the polypeptide chain being made (see figure on the right).
5. The chain must be elongated by one a -amino acid unit at a time. This means one linking reaction for each a -amino acid in the chain, followed by separation of the products. Each reaction must, therefore, have a high yield and each purification step must separate most of the product.
Chemists solve the problems above as follows. Problems 1 and 2 by using a coupling reagent that increases the reactivity of the - COOH group under mild conditions.

Problem 3 by protecting reactive side chains with blocking groups that they can remove under mild conditions when all the links are complete.

Problem 4 by blocking the a -amino acid and polypeptide groups that they do not want to join, again using groups they can remove easily later.

Problem 5 by attaching the growing polypeptide chain to an insoluble support made from a resin. They then wash away soluble by-products and unused reagents before adding the next a -amino acid to the chain. When the synthesis is complete the polypeptide is detached from the resin.

diagram of peptide synthesis
Figure 1a
Solid state peptide synthesis.
The 1984 Nobel Prize for Chemistry was awarded to Professor Merrifield of Rockefeller University for developing an automated version of this technique capable of producing the hormone insulin (51 a -amino acids). Figure 1a summarises the sequence of steps involved. Figure 1b gives the primary structure of the two chains in human insulin. Figure 2 on page 1 shows the molecule in 3D.
Figure 1b
The primary structure of the two polypeptide chains in human insulin.

How many different tripeptides (three a -amino acids linked together) could you make using three different a -amino acids?

Unilever Education Advanced Series: Proteins
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