17β‐H YDROXYSTEROID D EHYDROGENASE TYPE 12
III.2.1. E XPRESSION OF HUMAN HSD17B12 IN P ICHIA P ASTORIS
( P.
PASTORIS)
The recombinant expression of human HSD17B12 followed by protein purification was aimed to clarify the biological functions of human 17β‐HSD12 in vitro. Since 17β‐HSD12 is classified as transmembrane protein colocalizing with the endoplasmic reticulum (ER), a eukaryotic expression system, namely the P. pastoris strains KM71 and X33, was chosen.
For recombinant expression, the coding sequence of the human HSD17B12 combined with a C‐terminally fused TEV protease recognition site was cloned into the yeast expression vector pPICZ‐A. As a result, the generated pPICZ‐A‐HSD17B12 plasmid construct encodes for the human 17β‐HSD12 protein fused to a C‐terminal His6 tag which can be cleaved by the TEV protease [Figure IV‐4]. The PCR analysis of either crude cell lysates or isolated genomic DNA from different P. pastoris clones using the AOX1 primer pair (# 2282 + # 2283) after the electroporation with pPICZ‐A‐HSD17B12 showed that only one out of ten potential positive clones of the P. pastoris KM71 and X33 strains recombined the linearized plasmid into the genome [data not shown]. Western blot analysis and enzymatic activity assays analyzing the reduction of estrone (E1) into 17β‐estradiol (E2) with P. pastoris KM71 – pPICZ‐A‐HSD17B12 or P. pastoris X33 – pPICZ‐A‐HSD17B12 expression cultures showed that both strains expressed enzymatically active 17β‐HSD12 [Table III‐5, Figure III‐29, Figure III‐30]. Though, both P. pastoris strains expressed only little amounts of His6‐tagged 17β‐HSD12 which were not obviously visible by Coomassie staining [Figure III‐29A]. Despite the generally low expression, it seemed that the expression was more efficient in the P. pastoris KM71 strain compared with the wild type P. pastoris strain X33. However, up to now, HSD17B12 could only be successfully expressed in cultures of up to 100 ml, whereas the upscaling of expression cultures resulted in a strongly reduced or absent expression of HSD17B12 from pPICZ‐A‐HSD17B12.
Table III‐5: Enzymatic activities of P. pastoris – pPICZ‐A‐HSD17B12 expression cultures.
P. pastoris E1 E2
in [%]
KM71 < 1
KM71 pPICZ‐A‐HSD17B12 81.8
X33 pPICZ‐A‐HSD17B12 8.5
Activity tests were carried out in reactions containing cell pellets of 2 ml 48 h expression cultures (OD600 ≈ 10), 50 nM 3H‐labeled estrone, and 300 mM NADPH in reaction buffer. Given is the conversion of estrone (E1) into 17β‐estradiol (E2) in reactions after an incubation at 37 °C for 5 h.
III.2.2. S
OLUBILIZATION OF HUMAN17 β‐HSD12
FROMP.
PASTORIS EXPRESSION CULTURESWestern blot analysis of fractions resulting from the lysis of cell pellets from P. pastoris X33 – pPICZ‐A‐HSD17B12 and P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression cultures without the addition of any detergent showed that the overexpressed 17β‐HSD12 was partly found as “soluble” protein in the supernatant fraction of lysates, thereby, possessing the anticipated molecular weight of the His6‐tagged protein (38.3 kDa) [Figure III‐29B, Figure III‐30A]. Up to now, it has been unclear whether 17β‐HSD12 was appeared in the supernatant fraction as soluble protein or implemented in microsomes. However, as expected for membrane proteins, the majority of 17β‐HSD12 was found as non‐soluble protein in the pellet fractions.
For any reasons, beside the protein band of anticipated molecular weight, a second His6‐ tagged 17β‐HSD12 band with lower molecular weight (approx. 31 kDa) showed up in the pellet fraction of lysates, too [Figure III‐29B, Figure III‐30A]. Whereas no 17β‐HSD12 was detectable in the supernatant after the spheroblasting process, washing of spheroblasts with 1 M sorbitol lead to some release of soluble 17β‐HSD12 into the wash fraction [Figure III‐29].
Figure III‐29: Little amounts of “soluble” His6‐tagged 17β‐HSD12 can be detected in the supernatants of P. pastoris X33 – pPICZ‐A‐HSD17B12 expression culture lysates via Western blotting.
Shown are exemplary results from the detection of His6‐tagged 17β‐HSD12 in fractions resulting from the lysis of P. pastoris X33 – pPICZ‐A‐HSD17B12 expression cultures in MilliQ‐H2O+PI (according to II.5.2.1) via Coomassie staining and Western blotting.
(A) Coomassie stained SDS‐PAGE mini gel with equal volumes of fraction samples loaded. (B) Western blot with equal volumes of fractions using mouse‐anti‐His (1:4000) and HRP‐conjugated goat‐anti‐mouse (1:10000) antibodies.
P, pellet (resuspended in 2 ml); SN, supernatant (10 ml); wash, supernatant from the washing step (10 ml).
As mentioned above, when the spheroblasts were resuspended in MilliQ‐H2O and lysed via freeze‐thaw cycles His6‐tagged 17β‐HSD12 was already detectable in the soluble supernatant fraction of P. pastoris – pPICZ‐A‐HSD17B12 expression culture spheroblasts. Using P. pastoris KM71 – pPICZ‐A‐HSD17B12 spheroblasts it was additionally seen that these supernatants contained enzymatically active 17β‐HSD12 by catalyzing the reduction of estrone (E1) in enzymatic activity assays with 3H‐labeled steroids [Figure III‐30A, B].
However, since the majority of His6‐tagged 17β‐HSD12 was found to appear as non‐soluble protein when overexpressed in P. pastoris, several non‐ionic detergents were tested in respect of their potential for increasing the amount of soluble and enzymatically active 17β‐HSD12.
Figure III‐30: The non‐ionic detergent Brij‐35 is able to solubilize enzymatically active 17β‐HSD12.
Shown are results from Western blotting and enzymatic activity assays analyzing the solubilization of enzymatically active 17β‐HSD12 with different detergents. For this, equal volumes of supernatant and pellet (resuspended in a comparable volume buffer) samples, resulting from solubilization experiments with pellets of P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression cultures (as described in II.5.2.1), were analyzed via Western blotting (using the mouse‐anti‐His (1:5000) primary and HRP‐conjugated goat‐
anti‐mouse (1:15000) secondary antibody pair) and enzymatic activity assays (using 20 nM 3H‐labeled estrone as substrate and 300 μM NADPH as cofactor).
(A, B) Western blot (A) and enzymatic activities (reduction of estrone within 5 h) (B) with fractions from the simultaneous lysis and solubilization of spheroblasts in absence (MilliQ‐H2O) and in presence of 2 % NP‐40 detergent (2 % NP‐40). The analyzed fractions resulted from a final centrifugation step at 450 x g and 4 °C for 2 min. (C, D) Western blot (C) and enzymatic activities (reduction of estrone within 6 h) (D) with fractions from the successive lysis and solubilization of spheroblasts. The analyzed fractions resulted from the solubilization of spheroblast lysate pellets (after ultracentrifugation) using different detergents and a final ultracentrifugation step at 55000 x g and 4 °C for 30 min.
E1, estrone; E2, 17β‐estradiol; maltoside, dodecyl‐β‐D‐maltoside; pyranoside, 2,6‐dimethyl‐4‐heptyl‐
β‐D‐maltopyranoside; P, pellet; SN, supernatant.
A first experiment examined the solubilization properties of the detergent NP‐40. Although the use of 2 % NP‐40 solution within the spheroblasts lysis step promoted the solubilization
of His6‐tagged 17β‐HSD12 (especially those fragments of smaller molecular weight), the
resulting supernatants were only marginally enzymatically active [Figure III‐30A, B]. This means that 2 % NP‐40 in MilliQ‐H2O was not suitable for the solubilization of His6‐tagged 17β‐HSD12 from P. pastoris expression cultures because it destroyed the enzymatic activity of solubilized 17β‐HSD12.
In order to identify detergents which mediate the solubilization of enzymatically active
His6‐tagged 17β‐HSD12, pellets resulting from spheroblast lysates of P. pastoris KM71 –
pPICZ‐A‐HSD17B12 expression cultures were resuspended, split, and treated with either 1 % Tween20R, 1 % dodecyl‐β‐D‐maltoside (maltoside), 1 % Brij‐35, or 1 % 2,6‐dimethyl‐4‐
heptyl‐β‐D‐maltopyranoside (pyranoside) in solubilization buffer [II.5.2.1].
Here, it was found that all four non‐ionic detergents possessed the potential to solubilize 17β‐HSD12 by showing similar full length 17β‐HSD12 signal intensities in both the resulting soluble supernatant fraction and the remaining insoluble pellet fraction in Western blots [Figure III‐30C]. However, although the initial spheroblast lysate pellet was enzymatically active (approx. 12 % E1 E2 conversion) in activity assays with 3H‐labeled estrone (E1) as substrate, no enzymatic activity was detectable in the supernatant and pellet fractions of samples treated with dodecyl‐β‐D‐maltoside or 2,6‐dimethyl‐4‐heptyl‐β‐D‐maltopyranoside [Figure III‐30D]. The pellet fractions resulting from the solubilization of 17β‐HSD12 from the spheroblast lysate pellets with Tween20R or Brij‐35 were able to catalyze the reduction of estrone (approx. 8 % and 7 % E1 E2 conversion, respectively) [Figure III‐30D]. The same was true for the supernatant fraction of the pellet treated with Brij‐35, which revealed a clear traceable but very low (about 1.5 % E1 E2 conversion) enzymatic activity [Figure III‐30D]
This low activity was in accord with the little amount of the full length 17β‐HSD12 protein in the fraction, already detected by Western blotting [Figure III‐30C]. In contrast, no clear enzymatic activity was seen in the supernatant fraction of Tween20R treated pellets, indicating that, despite showing good solubilization properties, Tween20R negatively affected the enzymatic activity of solubilized 17β‐HSD12 [Figure III‐30D].
The additionally tested detergents Anameg‐7, MEGA‐8, n‐octyl‐β‐D‐glucopyranoside, and sodium cholate showed no clear solubilizing properties [data not shown].
All in all, the four detergents 2,6‐dimethyl‐4‐heptyl‐β‐D‐maltopyranoside, dodecyl‐β‐D‐
maltoside, Brij‐35, and Tween20R revealed good solubilization capabilities for 17β‐HSD12 from pellets of spheroblast lysates. Tween20R and Brij‐35 reduced the overall enzymatic activity of the resulting pellets and supernatants with estrone by approximately 20‐25 %, whereas 2,6‐dimethyl‐4‐heptyl‐β‐D‐maltopyranoside and dodecyl‐β‐D‐maltoside abolished the activity of 17β‐HSD12 completely. Despite the fact that only the supernatant of Brij‐35 treated pellets catalyzed the reduction of estrone and that Tween20R seemed to impair the enzymatic activity of solubilized 17β‐HSD12, both detergents, Tween20R and Brij‐35, were further analyzed in terms of solubilizing enzymatically active 17β‐HSD12.
III.2.3. P
URIFICATION OF HUMAN17 β‐HSD12
FROMP.
PASTORIS EXPRESSION CULTURES VIA THEH
IS6 TAGFirst tests revealed that little amounts of enzymatically active 17β‐HSD12 were detectable in the supernatant of centrifuged lysates of P. pastoris – pPICZ‐A‐HSD17B12 expression culture spheroblasts, although no detergent was added [Figure III‐29, Figure III‐30].
Because of this 17β‐HSD12 was initially purified via its fused His6 tag without the addition of any detergent (see II.5.2.2 ‐ method A) from the supernatant of 17β‐HSD12 overexpressing P. pastoris KM71 cell lysates (+) after ultracentrifugation (UC1‐SN) [Figure III‐31]. In Western blots using the mouse‐anti‐His primary and HRP‐conjugated goat‐anti‐mouse secondary antibody pair as well as in enzymatic activity assays using 3H‐labeled estrone (E1) as substrate it was seen that enzymatically active full length His6‐tagged 17β‐HSD12 (molecular size approx. 38 kDa) bound to the HisTrap HP column since no clear His6 tag signals and enzymatic activities were detectable in the flow‐through (FT) and wash (W) fractions [Figure III‐31]. The elution with 300 mM imidazole revealed that the majority of full length 17β‐HSD12 was found in the first (EL1) and second (EL2) elution fraction [Figure III‐31A].
Proportional to the full length His6‐tagged 17β‐HSD12 protein amount detected in the Western blot fractions, the enzymatic activity was by far higher in the EL2 fraction (53.6 % E1 E2 conversion) when compared with the EL1 fraction (7.8 % E1 E2 conversion) [Figure III‐31B]. Although weak signals corresponding to the size of full length His6‐tagged 17β‐HSD12 were also visible in the third (EL3) and fourth (EL4) elution fraction, no enzymatic activity was measureable in those fractions [Figure III‐31]. In addition to the full length His6‐ tagged 17β‐HSD12, a more prominent second His6‐tagged protein signal at approximately 20 kDa was detectable in the second to fourth elution fraction [Figure III‐31A]. Since this signal band was missing in the purified supernatant (UC1‐SN) but was present in the P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression culture lysate (total) and the pellets of the ultracentrifuged lysates (UC1‐P), the occurring signal band might represent an insoluble and inactive His6‐tagged 17β‐HSD12 degradation product [Figure III‐31A].
The solubilization of the lysate pellet resulting from the first ultracentrifugation step (UC1‐P) with either 1 % Brij‐35 or 1 % Tween20R which was followed by a second ultracentrifugation step for the separation of soluble and insoluble His6‐tagged 17β‐HSD12 showed that full length 17β‐HSD12 but also smaller fragments could be found in the supernatant (UC2‐SN) after the second ultracentrifugation step, though, the majority of full length His6‐tagged 17β‐HSD12 remained in the insoluble pellet fraction (UC2‐P) [Figure III‐31A]. Although the two detergents revealed no clear difference in their solubilizing capability, a huge difference was again seen in the enzymatic activity of supernatants resulting from Brij‐35 and Tween20R treated samples (UC2‐SN). Here, 27.6 % of estrone could be converted by the addition of the UC2 supernatant sample resulting from the Brij‐35 treated UC1 pellet, whereas the respective UC2 pellet as well as the supernatant and pellet of the Tween20R treated sample reduced only 3‐4 % of the estrone [Figure III‐31B].
The control samples from non‐transformed P. pastoris KM71 lysates (‐) did neither show signals in Western blots nor possessed enzymatic activity [Figure III‐31].
Figure III‐31: Enzymatically active His6‐tagged 17β‐HSD12 can be purified from the detergent‐free supernatant of ultracentrifuged P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression culture spheroblasts lysate and, by using the non‐ionic detergent Brij‐35, solubilized from the remaining pellet.
Shown are 17β‐HSD12 amounts and enzymatic activities in fractions resulting from the detergent‐free purification of His6‐tagged 17β‐HSD12 from supernatants of ultracentrifuged P. pastoris KM71 – pPICZ‐A‐
HSD17B12 lysates and its solubilization from the remaining ultracentrifugation pellet by using the non‐
ionic detergents Brij‐35 and Tween20R. Pellets from 50 ml P. pastoris KM71 – pPICZ‐A‐HSD17B12 48 h expression cultures were processed as described in II.5.2.2 ‐ method A and fractions from the purification as well as from the solubilization reactions were analyzed by Western blotting and enzymatic activity tests.
Non‐transformed P. pastoris KM71 cultures served as negative controls. Within each approach (purification and solubilization of 17β HSD12), equal sample volumes were applied in the analyses. (A) Results from Western blots with fractions using the mouse‐anti‐His (1:5000) primary and HRP‐conjugated goat‐anti‐
mouse (1:15000) secondary antibody pair. (B) Results from activity tests with fractions analyzing the reduction of 20 nM 3H‐labeled estrone in presence of 300 μM NADPH overnight (bars). The blue lines indicate the total volume of the respective fractions.
–, P. pastoris KM71 culture; +, P. pastoris KM71 – pPICZ‐A‐HSD17B12 48 h expression culture; E1, estrone;
E2, 17β‐estradiol; EL1, first elution fraction (1 ml); EL2, second elution fraction (1.5 ml); EL3, third elution fraction (1 ml); EL4, fourth elution fraction (3 ml); FT, flow‐through fraction (10 ml); n.a., not analyzed;
UC1‐P, pellet resulting from the first ultracentrifugation step (1 ml); UC1‐SN, supernatant resulting from the first ultracentrifugation step (10 ml); UC2‐P, pellet resulting from the second ultracentrifugation step (1 ml);
UC2‐SN, supernatant resulting from the second ultracentrifugation step (1 ml); wash, wash fraction (5 ml).
An optimization of the purification method aimed to augment the yield of purified and enzymatically active recombinant human 17β‐HSD12. Because it was seen previously that Brij‐35 is able to solubilize enzymatically active His6‐tagged 17β‐HSD12, two alternative procedures adding Brij‐35 to spheroblasts at a very early stage of purification were tested (see II.5.2.2 ‐ method B). These two Brij‐35 containing purification methods combined the detergent mediated lysis of spheroblasts and the solubilization of 17β‐HSD12 prior to the purification of His6‐tagged 17β‐HSD12 in order to increase the amount of 17β‐HSD12 in the soluble fraction of P. pastoris lysates and thus the yield of purified 17β‐HSD12 as well as to accelerate the process and thus reduce a possible loss in enzymatic activity. The capabilities of these Brij‐35 containing purification methods were again analyzed by Western blotting and enzymatic activity assays with fractions gained from the purification of His6‐tagged 17β‐HSD12 from P. pastoris KM71 – pPICZ‐A‐HSD17B12 55 h expression cultures by using Econo‐Pac columns with Ni Sepharose resin bed [Figure III‐32].
Figure III‐32: Enzymatically active His6‐tagged 17β‐HSD12 can be purified from P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression cultures by two alternative Brij‐35 containing purification procedures combining the lysis of spheroblasts and the solubilization of 17β‐HSD12.
Shown are 17β‐HSD12 amounts and enzymatic activities in fractions resulting from the purification of His6‐ tagged 17β‐HSD12 from P. pastoris KM71 – pPICZ‐A‐HSD17B12 expression cultures via two alternative Brij‐35 containing purification procedures, combining the lysis of spheroblasts and the solubilization of 17β‐HSD12. Pellets from 2x100 ml P. pastoris KM71 – pPICZ‐A‐HSD17B12 55 h expression cultures were processed as described in II.5.2.2 ‐ method B and equal volumes of fraction samples were analyzed by Western blotting and activity assays. (A) Results from Western blots with fractions using the rabbit‐anti‐His (1:3000) primary and HRP‐conjugated goat‐anti‐rabbit (1:5000) secondary antibody pair. (B) Results from enzymatic activity tests with fractions analyzing the reduction of 20 nM 3H‐labeled estrone in presence of 300 μM NADPH overnight (bars). The blue lines indicate the total volume of the respective fractions.
E1, estrone; E2, 17β‐estradiol; EL1, first elution fraction (2 ml); EL2, second elution fraction (2 ml); EL3, third elution fraction (2 ml); FT, flow‐through fraction (15 ml); marker, PageRuler Prestained Protein Ladder; SN, supernatant (15 ml); P, pellet (1 ml); W1, first wash fraction (2 ml); W2, second wash fraction (2 ml); W3, third wash fraction (2 ml).
The Western blots showed that generally only very weak full length His6‐tagged 17β‐HSD12 signals were detectable in the lysates and the supernatants (SN) after an ultracentrifugation step, whereas more intense signals were visible in the 15‐times concentrated pellet (P) fractions [Figure III‐32A]. In addition, clearly enriched full length His6‐tagged 17β‐HSD12 signals were seen in all elution fractions (EL1 to EL3) but not in the flow‐through (FT) and wash (W) fractions [Figure III‐32A]. The insertion of an extra centrifugation step at 40 x g and 4 °C for 3 min after the spheroblasting reaction revealed no obvious differences in the yield of 17β‐HSD12 on protein level as seen in Western blots [Figure III‐32A]. Though, it seemed that the intensity of signals at and below the molecular weight of full length His6‐tagged 17β‐HSD12 was a little bit higher in fractions resulting from the purification without the additional centrifugation step [Figure III‐32A]. However, looking at the enzymatic activity determined in the fractions with 3H‐labeled estrone (E1) as substrate, a clear difference was seen between the two procedures. Although not detectable in Western blotting, significant amounts of enzymatically active 17β‐HSD12 were found in the flow‐through (FT) and first wash (W1) fractions (15.4 % and 3.8 % E1 E2 conversion, respectively) using the procedure without an additional centrifugation step after the spheroblasting [Figure III‐32B]. Generally, the enzymatic activities found in the elution fractions corresponded to the intensities of full length His6‐tagged 17β‐HSD12 detected in Western blots [Figure III‐32]. In principle, elution fractions resulting from the purification without an additional centrifugation step after spheroblasting exhibited higher activities when compared with the respective fractions of the additionally centrifuged spheroblasts (46.3‐90.4 % versus 7.1‐75.3 % E1 E2 conversion) [Figure III‐32B]. Moreover, the extra centrifugation step seemed to result in losses in enzymatic activities measured in the supernatant and pellet fractions (12.3 % and 8.1 %
E1 E2 conversion, respectively, versus 39.2 % E1 E2 conversion in the lysate). Contrary,
the purification process of the additionally centrifuged lysates was easier because the plugging of the Ni Sepharose resin bed was reduced and it seemed to yield eluates with higher purity [data not shown]. All in all, by considering the total volumes of fractions, a clear but not closer ascertained enrichment of 17β‐HSD12 on protein and enzymatic activity level was seen in the elution fractions with both purification method variants.
In summary, it was possible to purify enzymatically active His6‐tagged 17β‐HSD12 from the P. pastoris expression system, however, with yet unsettled purity. Although the expression of pPICZ‐A‐HSD17B12 was slightly higher in the P. pastoris KM71 strain when compared with the wild type X33 strain, the overall expression level was very low and the protein product only visible via Western blotting. Different purification procedures showed that small amounts of 17β‐HSD12 occur in the soluble supernatant after ultracentrifugation and that the yield of soluble and enzymatically active 17β‐HSD12 could be increased by the addition of 1 % Brij‐35 for the lysis of spheroblasts. Up to now, the upscaling of small scale expression cultures and purification batches in order to produce and purify higher amounts of 17β‐HSD12, respectively, have not been successful. Thus, it was not possible to perform extensive activity assays with purified 17β‐HSD12 for its closer enzymatic characterization because the yields of 17β‐HSD12 from small‐scale approaches were too low.