A Simple and Reproducible Method to Isolate Plasmid DNA from Yeast after a Two-Hybrid...

Introduction

The yeast two-hybrid system is a widespread method used to study protein-protein interactions ^{(1) (2) (3)} . In this system, one protein, the "bait" molecule, is fused to a DNA-binding domain (e.g., Escherichia coli LexA protein), and the other partner, the "prey" molecule, is fused to an activation domain (e.g., yeast GAL4 protein). When these two hybrid proteins interact, a bipartite transcription factor is reconstituted and can transactivate reporter genes, such as lacZ (encoding beta-galactosidase) or his3 (encoding imidazole acetol phosphate transaminase enzyme), which are downstream of DNA-binding sites for the bait protein's DNA-binding domain. The system is also of great use for detecting and characterizing new binding partners for a specific protein that is fused to the DNA-binding domain. This is achieved by screening a library of cDNAs fused to the sequence of the activation domain. In a typical screening protocol, the plasmid DNA from each yeast clone must be isolated in order to identify the cDNA.

Here we describe a procedure to isolate high-quality plasmid DNA from yeast, suitable for PCR amplification and E. coli transformation. This method couples digestion with lyticase, an enzyme that hydrolyzes poly(beta-1,3-glucose) of the yeast cell wall, followed by the Wizard® Plus SV Minipreps DNA Purification System protocol. The total procedure is simple, reproducible and can be used routinely to isolate plasmid DNA from yeast. Lyticase digestion was preferred over disruption with glass beads because lyticase digestion is more reproducible and allows a higher yield of DNA.

Procedure

1. Collect yeast from a liquid culture (2ml) and resuspend in 50µl of lysis buffer (50mM Tris-HCl [pH 7.5], 1.2M sorbitol, 10mM EDTA and 10mM beta-mercaptoethanol added just before use). Alternatively, if the yeast clone has been spread on a plate in order to form an area of strong growth (typically a 2-3cm² patch), it can be collected with a sterile applicator or pipette tip. Mix vigorously by vortexing.
2. Digest the cell wall by adding 200 units of lyticase (30 units/µl, Sigma Cat.# L2524) to each tube. Incubate at 37°C overnight without shaking.
3. Collect yeast by centrifugation for 5 minutes at 4,000rpm. Remove and discard the supernatant.
4. Perform plasmid DNA isolation on the yeast pellet with the Wizard® Plus SV Minipreps DNA Purification System as described in Technical Bulletin #TB225 (see Section III.B, Step 2). (Note: The plasmid DNA will be in the spheroplast pellet at this point.)

Following isolation from the Saccharomyces cerevisiae strain L40*, the plasmid DNA was used as template in PCR amplification as shown in Figure 1. Purified plasmid can also be used to transform E. coli (1µl is typically used to transform electrocompetent HB101 strain; data not shown). To obtain enough yeast plasmid for restriction digestion analysis, transformation of E. coli followed by plasmid purification is necessary ⁽⁴⁾ .

Figure 1. Plasmid was isolated from yeast using the Wizard^® Plus SV Minipreps DNA Purification System.

Plasmid DNA was isolated as described in the text, and 1µl of the purified plasmid was used as template in PCR amplification using vector-specific primers (lanes 1–15). Due to the absence of plasmid exclusion in yeast, a single clone may contain several different plasmids. This can result in the amplification of multiple bands in the same sample. (Lanes M, DNA size markers.)