Diversification and specialization of HIV protease function during in vitro evolution.

Authors:
O'Loughlin TL, Greene DN, Matsumura I
Associated Labs:
Matsumura Lab (Emory University)
Mol Biol Evol. 2006 Apr . 23(4):764-72.
PMID: 16423863
Our goal is to understand how enzymes adapt to utilize novel substrates. We and others have shown that directed evolution tends to generate enzyme variants with broadened substrate specificity. Broad-specificity enzymes are generally deleterious to living cells, so this observed trend might be an artifact of the most commonly employed high throughput screens. Here, we demonstrate a more natural and effective screening strategy for directed evolution. The gene encoding model enzyme HIV protease was randomly mutated, and the resulting library was expressed in Escherichia coli cells to eliminate cytotoxic broad-specificity variants. The surviving variants were screened for clones with activity against a reporter enzyme. The wild-type human immunodeficiency virus type I protease (HIV PR) is cytotoxic and exhibits no detectable activity in reactions with beta-galactosidase (BGAL). In contrast, the selected variants were nontoxic and exhibited greater activity and specificity against BGAL than did the wild-type HIV PR in reactions with any substrate. A single round of whole gene random mutagenesis and conventional high-throughput screening does not usually effect complete inversions of substrate specificity. This suggests that a combination of positive and purifying selection engenders more rapid adaptation than positive selection alone.

Want to be recognized as an author of this publication?

Create your LabLife profile and tag yourself! You will also be able to tag other authors after you log into your account.

Create New Account

Login

What is LabLife?

LabLife is a collection of tools to help scientists organize, share and discover.

Learn more