DNA amount
II.5. W ORKING WITH PROTEINS
II.5.1. P URIFICATION OF HUMAN AKR1B15 FROM E. COLI
In order to have pure protein for, e.g., activity tests [II.8] or cofactor binding assays [II.5.4], both AKR1B15 isoforms were purified via the fused His6 tag from pellets of 1000 ml E. coli BL21(DE3) – pET28a(+)‐AKR1B15.1 or E. coli BL21(DE3) – pET28a(+)‐AKR1B15.2 overnight expression cultures (see II.1.5). The applied purification procedure is described below.
Firstly, the E. coli expression culture cell pellets were resuspended in an appropriate volume of ice‐cold lysis buffer and lysed via four 30 sec ultrasonication – 30 sec ice bath cycles using a VirSonic 475 sonicator (Virtis) combined with an ultrasonic converter CL4 (Virtis) at level 7.
The lysates were centrifuged at 13000 x g and 4 °C for 30 min and the supernatants were filtrated through sterile syringe filters with a 0.22 μm pore size before 10 % Triton X‐100 solution was added to reach a final concentration of 2 % Triton X‐100. Afterwards, the Triton X‐100 supplemented supernatants were subjected to an automated purification of
His6‐tagged proteins using a Profinia Affinity Chromatography Protein Purification System
(Bio‐Rad) in combination with a 1 ml Bio‐Scale Mini Profinity IMAC cartridge (Bio‐Rad) and a downstream 10 ml Bio‐Scale Mini Bio‐Gel P‐6 Desalting Cartridge (Bio‐Rad). The chromatographic purification was performed at 4 °C according to the preassigned Profina method “Native IMAC + Desalting” (Bio‐Rad). However, instead of the standard buffers recommended for the IMAC purification, a modified set of buffers (i.e., buffers containing the detergent N‐lauroylsarcosine) was used for the purification of AKR1B15 isoforms.
1x desalting buffer: 20 1
mM mM
KPi(pH 8.0) EDTA in MilliQ‐H2O
All buffers used for purification were sterile filtered (0.22 μm pore size).
N‐lauroylsarcosine was added after filtration.
II.5.2. P
URIFICATION OF HUMAN17 β‐HSD12
FROMP.
PASTORISTE S T O F D E T E R G E N T S F OR S O L U B I L I Z A T IO N
In order to find suitable solubilizing agents for the purification of human 17β‐HSD12 from P. pastoris, different detergents (Anameg‐7, Brij‐35, 2,6‐dimethyl‐4‐heptyl‐β‐D‐malto‐
pyranoside, dodecyl‐β‐D‐maltoside, MEGA‐8, n‐octyl‐β‐D‐glucopyranoside, NP‐40, sodium cholate, and Tween20R) were tested.
To test whether Anameg‐7, MEGA‐8, n‐octyl‐β‐D‐glucopyranoside, NP‐40, or sodium cholate are proper additives, pellets from P. pastoris – pPICZ‐A‐HSD17B12 expression cultures (see II.2.3) were resuspended in SAED buffer (1‐2 ml per OD600 of harvested culture) before 10 μl of lyticase solution (final lyticase concentration: 3‐7.5 μg/ml) were added for spheroblasting. The mixtures were incubated on a CAT RM 5 (CAT M. Zipperer) roller mixer at room temperature for 3 h and then centrifuged at 600 x g for 10 min. The spheroblast pellets were resuspended in 1 M sorbitol solution using the same volume as used for the cell pellets and divided into appropriate aliquots. After an centrifugation step at 600 x g for 10 min, the resulting spheroblast pellets were resuspended either in MilliQ‐H2O+PI, in NP‐40 solution, or in breaking buffer which was supplemented with 10 % detergent stock solution (from Solution Master Detergent Kit, Affimetrix). Here, the taken volumes were again comparable to the volumes of the initial cell suspension. The spheroblasts were lysed via three freeze‐thaw cycles using liquid nitrogen for freezing and lukewarm water for thawing.
Finally, the lysates were centrifuged at 450 x g and 4 °C for 2 min. Samples of all steps were separated by PAGE [II.5.5] and subjected to Western blotting [II.5.6] in order to monitor the presence of His6‐tagged 17β‐HSD12 in each fraction. In addition, supernatants from the last centrifugation step were frozen at ‐80 °C for storage and analyzed via activity tests with
3H‐labeled substrates [II.8.1] at a later date.
To test whether 2,6‐dimethyl‐4‐heptyl‐β‐D‐maltopyranoside, Brij‐35, dodecyl‐β‐D‐maltoside, or Tween20R are proper additives, pellets from P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression cultures (see II.2.3) were resuspended in 8 ml suspension buffer (0.8 ml per
OD600 of harvested culture) before adding 500 μl of lyticase solution for spheroblasting. The
mixtures were incubated on a CAT RM 5 (CAT M. Zipperer) roller mixer at room temperature for 1 h. The spheroblasts were subsequently lysed via two freeze‐thaw cycles (liquid nitrogen – lukewarm water) and two sonication‐ice cycles (15 sec ultrasonic bath – 15 sec ice bath) without any centrifugation step in between. The lysates were transferred into ultracentrifugation tubes (Beckman) and centrifuged at 125000 x g and 4 °C for 1 h using an OptimaMAX ultracentrifuge with MLA‐80 rotor (Beckman Coulter). The resulting pellets
were resuspended in 5 ml solubilization buffer and split into 900 μl aliquots. 100 μl of the respective 10 % detergent solution were added to the 900 μl aliquots (final detergent concentration 1 %) and incubated at 4 °C while rotating on a LD79 test‐tube rotator (Labinco) to solubilize membrane proteins, including the 17β‐HSD12. Afterwards, the mixtures were centrifuged in the OptimaMAX ultracentrifuge with TLA‐55 rotor (Beckman Coulter) at 55000 x g and 4 °C for 30 min. Samples of all steps were frozen at ‐80 °C until they were analyzed via PAGE [II.5.5] followed by Western blotting [II.5.6] and activity tests with
3H‐labeled substrates [II.8.1] at a later time point.
lyticase solution: 3 mg/ml lyticase
in MilliQ‐H2O
1 M sorbitol solution: 1 M sorbitol
in MilliQ‐H2O
MilliQ‐H2O+PI 1 tablet/10 ml cOmplete Mini, EDTA‐free Protease
Inhibitor Cocktail (Roche)
10 % Brij‐35 solution: 10 % (w/v) Brij‐35
in solubilization buffer
10 % maltoside solution: 10 % (w/v) dodecyl‐β‐D‐maltoside
in solubilization buffer
10 % pyranoside solution: 10 % (w/v) 2,6‐dimethyl‐4‐heptyl‐β‐D‐
maltopyranoside in solubilization buffer
10 % Tween20R solution: 10 % (v/v) Tween20R
in solubilization buffer
PR E L I M I NA R Y P U R I F I C A T I O N M E T H O D S F O R 17β‐HSD12
Two widely differing methods were used for the small‐scale purification of His6‐tagged 17β‐HSD12 from P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression cultures (see II.2.3).
Method A describes the detergent‐free purification of 17β‐HSD12 from supernatants of ultracentrifuged cell lysates and the solubilization of active 17β‐HSD12 from the remaining pellets. In contrast, method B combines the lysis of spheroblasts and the solubilization of 17β‐HSD12 through the addition of Brij‐35 prior to the purification.
METHOD A: DETERGENT‐FREE 17β‐HSD12 PURIFICATION METHOD
Cell pellets resulting from 50 ml expression cultures (OD600: 5‐10) were resuspended in 10 ml suspension buffer before 500 μl lyticase solution were added for spheroblasting.
The mixtures were incubated on a CAT RM 5 (CAT M. Zipperer) roller mixer at room temperature for 1 h and the spheroblasts were subsequently lysed via two freeze‐thaw cycles (liquid nitrogen – lukewarm water) and three sonication‐ice cycles (15 sec ultrasonic bath – 15 sec ice bath) without any centrifugation step in between. To reduce the presence of cell debris or nuclei in the subsequent ultracentrifugation and solubilization steps, the cell lysates were at first centrifuged at 500 x g and 4 °C for 2 min before the resulting supernatants were transferred into ultracentrifugation tubes (Beckman Coulter) and centrifuged at 125000 x g and 4 °C for 1 h using an OptimaMAX ultracentrifuge with MLA‐80 rotor (Beckman Coulter). The supernatants were taken, supplemented with 2 M imidazole solution to reach a final concentration of 20 mM, and manually loaded onto a 1 ml HisTrap HP column (GE Healthcare Life Sciences). The loaded columns were washed with 5 column volumes 20 mM wash buffer to reduce weak bound proteins before His6‐tagged 17β‐HSD12 was eluted with 300 mM elution buffer in fractions of 1 ml, 1.5 ml, 1 ml, and 3 ml. In order to solubilize 17β‐HSD12 from the pellet of the first ultracentrifugation step, pellets were resuspended in either 1 ml 1 % Brij‐35 or 1 ml 1 % Tween20R suspension solution and afterwards incubated on a LD79 test‐tube rotator (Labinco) at 4 °C for at least 60 min. The suspensions were once more centrifuged using the OptimaMAX ultracentrifuge with TLA‐55 rotor at 55000 x g and 4 °C for 30 min and analyzed for the solubilization of His6‐tagged 17β‐HSD12 from the pellet.
METHOD B: BRIJ‐35 CONTAINING PURIFICATION METHOD
Cell pellets resulting from 100 ml expression cultures (OD600: 15) were resuspended in 15 ml suspension buffer before 1650 μl lyticase solution were added for spheroblasting.
These mixtures were incubated on a CAT RM 5 (CAT M. Zipperer) roller mixer at room temperature for 1 h without any lysis step afterwards. The spheroblast suspensions were mixed with 10 % Brij‐35 solution to reach a final concentration of 1 % Brij‐35 and then incubated on a LD79 test‐tube rotator (Labinco) at 4 °C for at least 60 min in order to lyse spheroblasts and solubilize 17β‐HSD12. Alternatively, prior to the addition of Brij‐35, the spheroblast suspensions were centrifuged at 40 x g and 4 °C for 3 min and the supernatants, including the spheroblasts, were taken. Finally, the solubilization reactions were centrifuged using an OptimaMAX ultracentrifuge with TLA‐55 rotor (Beckman Coulter) at 35500 x g and 4 °C for 1 h. For the purification of His6‐tagged 17β‐HSD12, the resulting supernatants were supplemented with 2 M imidazole solution to reach a final concentration of 20 mM and loaded onto a 1 ml Ni Sepharose High Performance (GE Healthcare Life Science) resin bed in an Econo‐Pac Chromatographic column (Bio‐Rad). The loaded resin was washed three times with 2 ml 20 mM wash buffer and once more with 6 ml 20 mM wash buffer before the bound
His6‐tagged 17β‐HSD12 was eluted three times with 2 ml 300 mM elution buffer.
All fractions gained from the purification and solubilization procedure were collected and subjected to activity tests with 3H‐labeled estrone [II.8.1] and PAGE [II.5.5] followed by Western blotting [II.5.6].
lyticase solution: 3 mg/ml lyticase
in MilliQ‐H2O
2 M imidazole solution: 2 M imidazole
in MilliQ‐H2O
10 % Brij‐35 solution: 10 % (w/v) Brij‐35
in MilliQ‐H2O