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GENERATION AND ISOLATION OF MODIFIED YEAST STRAINS TO ENHANCE RECOMBINANT INTEGRAL MEMBRANE PROTEIN EXPRESSION

IP.com Disclosure Number: IPCOM000241117D
Publication Date: 2015-Mar-27
Document File: 4 page(s) / 82K

Publishing Venue

The IP.com Prior Art Database

Abstract

This disclosure introduces a new approach to developing a yeast expression system capable of over-producing integral membrane proteins (IMP) from any organism. The system can include modified yeast expression strains specifically selected for IMP over-expression. The method described is based upon a set of yeast expression plasmids/constructs that permit quick cloning of target IMPs, straightforward culture optimization and subsequent protein purification of integral membrane proteins from any organism, including but not limited to human, animal, plant, bacterial, or insect. The IMPs may be natural or synthetic.

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Generation and Isolation of MODIFIED yeast strains to enhance recombinant integral membrane protein expression

Abstract

This disclosure introduces a new approach to developing a yeast expression system capable of over-producing integral membrane proteins (IMP) from any organism.  The system can include modified yeast expression strains specifically selected for IMP over-expression. The method described is based upon a set of yeast expression plasmids/constructs that permit quick cloning of target IMPs, straightforward culture optimization and subsequent protein purification of integral membrane proteins from any organism, including but not limited to human, animal, plant, bacterial, or insect.  The IMPs may be natural or synthetic.

BACKGROUND

Integral membrane proteins (IMPs) are a class of protein that includes channels, transporters, receptors, and enzymes and have a vital role in various cellular processes.  Researchers have attempted to express several of these proteins in prokaryotic expression systems, but found the proteins were either not expressed or formed insoluble, non-functional inclusion bodies.  Insect and mammalian cells have been used successfully to express small amounts of some of these IMPs, but costs and scalability can be problematic.

It has been demonstrated that yeast are amenable to eukaryotic IMP production.  Many of the currently available eukaryotic IMP structures were obtained from proteins produced heterologously in yeast cells.  In addition to its ease of manipulation, yeast allows for posttranslational modifications and proper membrane targeting and insertion, which are critical for functional IMP production.  However, most eukaryotic IMPs cannot be expressed in yeast systems at reasonable levels.  The greatest hurdle is the lack of knowledge about the intricate cellular processes to produce IMPs in the host system.  The conventional ways to optimize IMP over-expression rely on changing promoters, fusion tags, culture conditions (e.g., pH, temperature, aeration), or co-expressing chaperons.  These approaches require repeated rounds of time consuming, trial-and-error optimization, which is often protein specific. 

The modified yeast expression system is unique as it combines the advantages that yeast generally offers with the power of random mutagenesis/selection to overcome the low IMP expression issues.

METHODS (refer to Figure 1)

Vector construction:

One or more IMP construct may be used in this approach and can include selection constructs, expression constructs, or purification constructs.  An IMP expression construct includes a promoter functional in yeast to drive expression of a polynucleotide encoding a target IMP or fragment thereof.   In some cases, the promoter is an inducible or constitutive promoter.  The construct may include a selectable marker, a screenable marker, a purification tag or any combination thereof.   The IMP expression constructs used in the various steps may be con...