Engineered Enzymes

Abstract

The term ‘engineered enzymes’ encompasses the process of creating new proteins through manipulation of the DNA of the gene encoding the protein. The engineered gene is translated into protein through expression in a suitable cellular system.

Keywords: site‐specific mutagenesis; protein expression; recombinant enzymes; active site interaction; protein structure–function

Figure 1.

Site‐directed mutagenesis scheme. (a) PCR‐directed mutagenesis. Multiple rounds of the PCR are performed. In step 1, two separate sets of reactions amplify the plasmid sequence between primers A and B (1(i)) and primers C and D (1(ii)). Purified fragments from reactions 1(i) and 1(ii) are combined in another reaction using the external primers A and D, which anneal to only half of the possible combinations of overlapping fragments (2). Final products should contain the mutation (♦) in both strands (3). (b) Stratagene Quik‐ChangeTM method. Double‐stranded plasmid DNA is denatured (1), and two primers, complementary in sequence, are annealed to opposite strands of the template (1). Second strand synthesis is completed by Pfu polymerase (2). Treatment with DpnI endonuclease should digest any methylated DNA. The mutation (♦) should be on both strands of plasmids.

Figure 2.

(a) α‐Helical segment showing the radial distribution of amino acid side‐chains. The backbone is represented by a shaded ribbon, and the atoms are represented in capped sticks format. (b) β Hairpin. The up–down relationship of the amino acid side‐chains in succeeding positions in each strand can be seen in this diagram. The atoms are displayed in capped sticks format.

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References

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Further Reading

DeGrado WF (2001) Introduction: protein design. Chemical Reviews 101: 3025–3026 (Issue #10 of Volume 101 is devoted to Protein Design).

Penning TM and Jez JM (2001) Enzyme redesign. Chemical Reviews 101: 3027–3046.

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Regan L (1994) Born to be beta. Current Biology 4: 656–658.

Steipe B (1999) Evolutionary approaches to protein engineering. Current Topics in Microbiology 243: 55–86.

Schechter I and Berger A (1967) On the size of the active site in proteases. I. Papain. Biochemical and Biophysical Research Communications 27: 157–162.

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How to Cite close
Dunn, Ben M(Sep 2005) Engineered Enzymes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003896]