EUKARYOTIC HIGH RATE MUTAGENESIS SYSTEM

Although mutations are, in general, detrimental to organisms, there are situations in which a high mutation rate (hypermutation) is advantageous. For example, at the immunoglobin loci, hypermutation functions to improve antibody diversity and affinity to help the immune system cope with the large diversity of threatening microorganisms, which is itself mutational in origin.

To date, there are several methods to modify a given protein by mutagenesis, including site-directed, saturation, and scanner mutagenesis. Whatever the method of generating the mutants, the function of the individual mutants is subsequently assessed by transfecting individual mutant genes into the cells that are used for the biological read-out. In order to find an interesting mutant, one has to make an educated guess of where to modify the protein or investigate the properties of a large library of mutants.

To improve mutagenesis studies, UCSF researchers have developed a method for performing random saturation mutagenesis on target genes within a eukaryotic cell with minimal technical effort. The method exploits the immunoglobin hypermutation system that, when coupled to a selection system, will yield the protein variant of interest without prior insight into what the structural modifications should be. Briefly, a target gene is cloned into a vector containing enhancer fragments which effect hypermutation, and the construct is transfected into a mutator cell, of any cell lineage, whereupon hypermutation over the length of the target gene occurs.

The advantages of this method over current in vitro techniques are:

• High-speed random saturation mutagenesis with one-step cloning for mutagenesis and expression.
• Immediately delivers a “final” protein variant already properly processed (i.e. post-translational modifications, e.g. glycosylation).
• Can be coupled with selection system of choice that presents all the possible mutants due to the high mutation rate (10-4/bp/cell generation)

Intellectual Property:

UCSF holds issued U.S. Patent No. 5,885,827 claiming compositions for saturation mutagenesis using immunoglobulin hypermutation system. This patent is available for licensing.

If you would like to receive further information about this technology and potential licensing opportunities, please contact:

If you would like to receive further information about this technology and potential licensing opportunities, please contact:

Sunita Rajdev, Ph.D.
Licensing Officer
(415) 353-4470 phone
(415) 348-1579 fax
Sunita Rajdev

Reference: OTM Case #SF97-059