Working with yeast

2.2.7.1 Yeast-two-Hybrid methods

All methods in the following section were adapted from methods previously described by Uhrig and McFarlane (Uhrig and MacFarlane 2008).

2.2.7.1.1 Cultivation and maintenance of yeast (Saccharomyces cerevisiae)

Yeast colonies were grown for proliferation either on solid YPAD medium ( 2.1.11.4) at 30 °C or in liquid YPAD at 30 °C and 250 rpm.

2.2.7.1.2 Yeast (double) transformation

Transformation of yeast with both Yeast-two-Hybrid prey and bait plasmids was used to first test auto-activation of constructs and later confirm interaction with interaction candidates found in the Yeast-two-Hybrid screen. 10 ml liquid YPAD medium were inoculated with a freshly grown yeast AH109 colony and incubated overnight at 30 °C and 250 rpm. After incubation the OD600 of the cell suspension was measured and then used to inoculate 60 ml YPAD main culture to an OD600of 0.5.

The main culture was afterwards incubated at 30 °C and 250 rpm until the cell suspension reached an OD600of 2.0. The cells were subsequently harvested by centrifugation for 5 min at 4000 rpm. Following centrifugation, the supernatant was removed and the pelleted cells were resuspended in sterile 25 ml ddH2O. After centrifuging again for 5 min at 4000 rpm and discarding the supernatant, cells were resuspended in sterile 1 ml 100 mM lithium acetate and then transferred to 1.5 ml tubes. By centrifuging the tubes for 15 sec at 13 000 rpm, cells were once more pelleted. After discarding the supernatant, cells were resuspended in 100 µl 100 mM lithium acetate and subsequently aliquoted to 50 µl volumes. The aliquots were centrifuged in 1.5 ml tubes for 15 sec at 13 000 rpm, the supernatant

was discarded and 350 µl sterile transformation mix, containing 240 µl 50 % (w/v) PEG3350M, 36 µl 1M lithium acetate, 50 µl 2 mg/ml herring sperm DNA (heated to 96 °C for 10 min and then kept on ice prior to use) and 24 µl ddH2O, were added together with 12 µl plasmid mix containing 300 ng of each plasmid. This mixture was vortexed until the pelleted cells were resuspended and afterwards incubated for 30 min at 30 °C, followed by incubation at 42 °C for another 30 min. Prior to plating onto SD dropout plates, cells were pelleted via centrifugation for 15 sec at 6000 rpm and resuspended in 200 µl sterile ddH2O. Plates were incubated for 3 days at 30 °C.

2.2.7.1.3 Amplification of cDNA libraries for Yeast-two-Hybrid screen

Yeast-two-Hybrid screens are employed to discover potential interaction partners of proteins of interest. For this, the protein of interest is fused as bait to the Gal4 binding domain while cDNA libraries coding for potential interaction partners fused to the Gal4 activating domain act as prey. In this study, potential LSCE2-target proteins in Arabidopsis were searched for, so in order to get a preferably complete pool of Arabidopsis targets, two libraries, a standard Arabidopsis library from Arabidopsis cell suspension culture (Nemeth et al. 1998) as well as a root library (Klopffleisch et al.

2011) , were screened. Both libraries were obtained from J. Uhrig (Department of Plant Molecular Biology and Physiology, Göttingen University). Libraries consist of aliquots containing yeast cell suspensions where each cell carries one plasmid containing a prey construct. These aliquots are prepared by first resuspending cells from an already existing aliquot in 20 ml sterile ddH2O and plating 100 x 200 µl of this suspension onto SD -Leu plates (10 x 10 cm, 2.1.11.4) using sterile glass beads for spreading the cells evenly. In order to test viability and cell content, the cell titer of the suspension was determined by plating 10, 1 and 0.1 µl of it onto SD -Leu plates. After two days incubation at 30

°C, the colonies were washed off the SD -Leu plates with 10 ml YPAD each. The resulting cell suspension was collected in a 1 l Erlenmeyer flask and its OD600measured before it was incubated on ice for 1 h. After chilling, the suspension was transferred to 50 ml tubes and centrifuged at 4000 rpm for 5 min at 4 °C. After discarding the supernatant, cells were resuspended in 30 ml ice-cold YPAD and united in a 1 l Erlenmeyer flask. The OD600 of this suspension was then measured and ice-cold YPAD was added accordingly to reach an OD600of 40. 500 ml of the resulting suspension were mixed with 500 ml ice-cold, 50 % (v/v) glycerol, aliquoted and incubated at -20 °C for 30 min before being transferred to -80 °C.

2.2.7.1.4 Yeast-two-Hybrid screen: interaction mating and selection

Yeast AH109 colonies transformed with bait constructs (2.2.7.1.2) and grown on YPAD plates were used to inoculate 50 ml SD (-Trp, 4 % Glucose, 2.1.11.4). The liquid culture was incubated overnight at 30 °C, shaking at 200 rpm. The next day, prey libraries (2.2.7.1.3) were quickly thawed by applying body heat and used to inoculate 200 ml YPAD (2.1.11.4) in 1 l Erlenmeyer flasks. The suspension was incubated 1 h at 30 °C and 200 rpm before the OD600of both prey and bait cultures were measured.

The volume equivalent of 10 OD from both bait and prey culture were mixed and subsequently centrifuged for 4 min at 4000 rpm (RT). After discarding the supernatant cells were resuspended in 10 ml YPAD containing 20 % PEG6000 and transferred to 100 ml flasks. The cell suspensions were then incubated for 4.5 h at 30 °C and 80 rpm during which mating between yeast cells containing prey and bait constructs took place. The suspensions were then centrifuged for 4 min at 4000 rpm, the supernatant was discarded and cells were resuspended in 2 ml SD (-Leu, -His, -Trp). In order to determine mating efficiency, 10, 20 and 50 µl of the 2 ml cell suspension were spread onto SD (Leu, -His) plates and incubated 2 days at 30 °C. The suspension was then mixed with 500 ml semi-solid SD medium (-Leu, -His, -Trp, 2.1.11.4) by vigorous shaking. This selection mix was afterwards poured into 10 – 15 Ø 10 cm petri dishes and left to incubate for 5 – 10 days until colonies had grown.

2.2.7.2 Expression of LSCE2 and LSCE2-like inPichia pastoris

The following methods are based on the protocol included in the Easy Select Pichia Expression Kit (Thermo Fisher Scientific, Waltham, MA, USA).

2.2.7.2.1 Cultivation and maintenance of P. pastoris

Pichia was grown for proliferation either in liquid YPD (2.1.11.5) at 30 °C shaking at 200 rpm or on solid YPD plates at 28 °C.

2.2.7.2.2 Preparing P. pastoris cells for electroporation

Cells for electroporation were prepared by first inoculating 2 x 2.5 ml YPD with freshly grown P.

pastoris X-33 colonies. This pre-culture was incubated at 30 °C and 160 rpm. After around 8 hours,

°C and 160 rpm. The next day, the OD600 of the liquid culture was measured and subsequently adjusted to 0.3 before it was again incubated as described above until an OD600 of 1.2 – 1.5 was reached. Then, the liquid cultures were centrifuged in 2 x 500 ml centrifuge bottles at 2 000 g and 4 °C for 5 min. After discarding the supernatant, pellets were resuspended and combined in 100 ml YPD, mixed with 20 ml 1 M HEPES pH 8 ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)). Following resuspension, 2.5 ml 1 M DTT (dithiothreitol) were mixed gently into the suspension which was then incubated for 15 min at 30 °C. After incubation, the suspension volume was adjusted to 400 ml with ice-cold ddH2O. The suspension was then centrifuged at 2 000 g and 4 °C for 5 min and the cell pellet resuspended in 250 ml ice-cold ddH2O. Following another centrifugation step (2 000 g, 4 °C, 5 min), cells were resuspended first in 20 ml 1 M ice-cold sorbitol and then after repeating the centrifugation in 500 µl 1 M ice-cold sorbitol. From this suspension, 80 µl aliquots were taken and used directly for electroporation (see below).

2.2.7.2.3 Electroporation of P. pastoris cells

80 µlP. pastoris cells (see above) were mixed with 10 µl linearized (digested with restriction enzyme PmeI) plasmid DNA. The mixture was transferred to a pre-cooled electroporation cuvette (2 mm cuvette gap width, Biozym; Hessisch Oldendorf, Germany) and kept on ice for 5 min. Electroporation was performed with the Micro Pulser™ (BioRad, Munich, Germany), with a pulse strength of 2 kV.

Immediately after electroporation, 1 ml ice-cold 1 M sorbitol was added to the cell suspension which was subsequently transferred to a 15 ml tube and incubated 1 – 2 h at 30 °C. Cells were then spread in 80, 100 and 150 µl aliquots onto YPD plates containing 100 or 500 µg/ml zeocin. Plates were incubated 3 days at 28 °C.

2.2.7.2.4 Determination of Mut-phenotype of P. pastoris transformants

P. pastoris strain X-33 is Mut⁺, meaning that, due to the fact that the AOX1 gene is not disrupted (which is the case in Mut^S strains), X-33 is able to grow effectively on medium containing methanol.

This was tested by picking 20 individual transformants and transferring a small amount of colony material with a sterile pipette tip to plates consisting of rich YPD medium containing 100 µg/ml zeocin, Minimal Dextrose Medium or Minimal Methanol Medium (2.1.11.5). Colonies were grown 3 days at 28

°C.

2.2.7.2.5 Small-scale expression of recombinant proteins in Pichia

Growth conditions for optimal protein expression and secretion by Pichia transformants vary for individual proteins. It is therefore necessary to determine the best temperature and medium methanol content at which transformants are grown. In a small-scale approach, five colonies were selected from YPD plates grown as described in 2.2.7.2.4 and added to 3 ml YPD containing 500 µg/ml zeocin. The liquid cultures were grown for around 7 hours at 30 °C, shaking at 210 rpm. 600 µl of these pre-cultures were used to inoculate 10 ml BMGY ( 2.1.11.5) which were then incubated at 30 °C and 210 rpm. After one and a half days (~ 32 h) of growth, cells were pelleted via centrifugation at 3 000 g for 15 min at room temperature and afterwards resuspended in BMMY containing 3 % methanol (2.1.11.5). The cell suspensions were incubated at either 16 or 25 °C, shaking at 210 rpm for two days.

Then, cell cultures were fed with 100 % methanol to a concentration in the culture of either 1 or 3 % each day for 5 days, resulting in a total 7 days of growth after inducing LSCE2/-like expression via methanol inPichia.