Artificial Minitransposon Used to Create Protein Mutation Expression Library

An efficient new method for producing mutated-protein expression libraries provides unique benefits for protein structure-function studies and for protein engineering.

The minitransposon was specifically designed and used, by random insertion via the MuA transposase enzyme, to create a gene library for expressing a set of protein mutants that provides distinct protein sequence diversity over existing methods.

Libraries expressing multiple mutants of a given protein are particularly useful for studying protein mechanisms and the adaptation of proteins during molecular evolution, as well as for designing proteins (such as biosensors and molecular switches) with novel functions in synthetic biology, including potential drug therapy enzymes.

One type of library in use is made up of "circularly permuted" protein mutants, proteins that have been mutated by connecting the two ends (the N- and C-termini) and later breaking elsewhere, often also having altered part(s) in between.

The new method, called PERMutation Using Transposase Engineering (PERMUTE), for making this type of library was developed by senior undergraduate student Manan Mehta and mentor Jonathan Silberg, assistant professor, at Rice University's Department of Biochemistry and Cell Biology (Houston, TX, USA), with assistance from research technician and coauthor Shirley Liu.

"Creating such a library has traditionally required painstaking processes," said Prof. Silberg. "Existing methods for rearranging the bits of information in a protein are slow and arduous to use," he said. "In addition, they are nonideal because they simultaneously create multiple types of mutations, the desired rearrangements, and undesired deletions of important amino acids.”

Prof. Silberg further explained, "With our method, you only generate mutants with rearranged sequences, and you don't need to be an expert in biomolecular engineering. All you need is the DNA that encodes your gene of interest, the artificial minitransposon we engineered and an enzyme. […] It's a way of making, with great control, all of the diversity of this type of mutation that could exist in a protein."

In this particular study, published online ahead of print on February 7, 2012, in the journal Nucleic Acids Research, a library was created where the enzyme adenylate kinase was mutated. The library of mutant proteins was then mined for variants that retained catalytic functional by introducing them into Escherichia coli bacteria then screening and selecting for adenylate kinase activity. Out of about 220 sequenced mutants (about half of which were active), 15 unique sequence variants were identified.

The work was supported by the National Aeronautics and Space Administration (NASA; USA) and the Robert A. Welch Foundation (The Welch Foundation; Houston, TX, USA).

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