Recombinant production of the nontruncated CYP52A13

The first exploratory experiments using CYPtr showed that the truncated enzyme remained bound to the microsomes and was not secreted to the medium. In the enzymatic assays, no conversion to the desired DCA occurred, nor was there any increased substrate-induced NADPH consumption. In the chimeric constructs, we observed CPR activity, and thus it was concluded that lack of activity is due to inactive CYPtr. It was also attempted to recombinantly produce CYP52A13 in a nontruncated form (CYPnt) in P. pastoris. In this way, it was investigated whether or not the truncations led to the loss of activity. Using a nontruncated form would additionally allow for the optimization of a self-sufficient chimer in its natural environment of the ER membrane, before continuing with solubilization efforts. Furthermore, a positive control could be created in this way.

CYPnt_pPICZA was linearized and transformed in P. pastoris NRRL-Y-11430. Six colonies were picked and tested in a small scale expression test. No Histag is present so not western blot but Coomassie staining of the SDS-PAGE gel was performed (Figure 3.28). As there are no truncations, CYPnt should reside in the microsomal fraction. To identify the gel band, coinciding with CYPnt, a microsome sample of the empty wild type P. pastoris strain was taken along. Comparing the profile of the six different colonies to the wild type microsome sample shows a different pattern between 50 and 75 kDa, the region where CYPnt (60 kDa) is expected.

Gel bands were cut out in this region and analysed by MALDI-TOF MS (data not shown). The more intense band around 75 kDa was identified as the peroxisomal alcohol oxidase 1 (AOX1).

Upon methanol induction, AOX1 can account for up to 30 % of the total soluble protein [312].

It is thus not surprising that AOX1 is contaminating isolated fractions. The two bands around 50 kDa were identified as the mitochondrial ATP synthase subunits α and β (ATP1 and ATP2).

The ATP synthase subunits are found in the inner membrane of the mitochondria and were

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reported to contaminate isolated microsomal fractions before [325]. The intermediate bands could not be identified. However, if CYPnt would be produced to a high yield, this should be visible as a prominent band in the 50-75 kDa region on gel. Efforts were done to improve the microsome isolation protocol in order to further enrich the microsomal proteins leading to the identification of recombinant CYPnt. Including additional wash steps of the microsomal fraction resulted in identification of several additional proteins in the aforementioned MW region, however, not leading to the identification of CYPnt (data not shown). Moreover, enzymatic cell lysis instead of mechanical cell lysis with glass beads was performed in order to leave peroxisomes and mitochondria intact and isolate these organelles using intermediate centrifugation speeds. Alternatively, a prolonged expression phase of 72 h and 96 h was executed in an attempt to increase the CYPnt yield. However, CYPnt could not be identified in any case (see Addendum).

Figure 3.28: SDS-PAGE analysis of 20 µg microsomal protein after small-scale expression test of six colonies picked after cprnt transformation in P. pastoris. Gel bands in the blue rectangle were cut out for MALDI-TOF MS.

CYPnt (60 kDa) was not identified in the analysed gel bands.

In parallel to the MALDI-TOF MS identification efforts, enzymatic assays were performed as an alternative way to prove production of an active CYPnt. At first, it was again assumed that the endogenous CPR from P. pastoris could serve as the alternative redox partner and it was tested whether whole cells could be used to assess activity instead of performing in vitro assays using isolated microsomes. On the one hand, the substrate oleic acid was added during the expression phase. On the other hand, cells were collected after the expression phase, washed and resuspended in phosphate buffer for a subsequent whole-cell bioconversion assay. After bioconversion, the products were extracted with diethyl ether and methylated for subsequent GC-MS analysis. Oleic acid was completely consumed after 24 h and no corresponding DCA appeared in the GC chromatogram (Figure 3.29). The same was observed in case of the in vitro whole-cell bioconversion assay, i.e. complete consumption of oleic acid without the appearance of DCA (data not shown). Although also 18-hydroxyoleic acid could be the final product, which is not detected with the applied GC-MS protocol, it is more likely that the oleic acid was completely consumed by β-oxidation in the peroxisomes. Upon methanol induction, it is known that the peroxisomes proliferate. Also FAs can be used as the sole carbon source and addition of FAs as well leads to peroxisome proliferation [326]. Therefore, further enzymatic assays were performed with isolated microsomes.

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Figure 3.29: GC chromatogram of samples taken during an in vivo bioconversion assay. Oleic acid was added to the P. pastoris culture during the cypnt expression phase and samples were taken every 12 h. Oleic acid was completely consumed after 24 h without DCA formation (expected RT = 27.45 min).

Microsomes were isolated and used in different enzymatic assays. First NADPH oxidation was monitored in presence and in absence of the substrate oleic acid. No difference in the rate of consumption was observed (data not shown). In another assay, the diethyl ether extract, collected after conversion, was silylated and analysed by GC-MS. No 18-hydroxyoleic acid, nor 1,18-octadecendioic acid was found in the chromatogram (data not shown). Of note, in both assays, recombinant CPRtr was added. As described before, the isolated CPRtr had a low activity per ml so it can be disputed whether this was enough to deliver the required electrons to CYPnt.

Using SDS-PAGE and subsequent MALDI-TOF MS of excised gel bands did not result in the identification of CYPnt. Also the performed enzymatic assays came back negative. Therefore, it was decided to add a Histag C-terminally to CYPnt enabling the use of the more sensitive western blot analysis. Additionally, it was asked whether the expression yield could be improved by altering the translation initiation sequence. Invitrogen proposes the following sequence as a suitable translation initiation sequence for high expression (G/A)NNATGG, that is the so-called Kozak sequence. First, the Histag-containing enzyme was constructed by PCR using back-to-back primers, followed by NheI digest and ligation. Back-to-back primers were then applied as well in order to modify the translation initiation sequence, resulting in the following sequence GAGATGGGT. A Gly residue was inserted between Met and Thr with the aim of introducing the guanine base following the ATG start codon. Three colonies were picked

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upon transformation of P. pastoris with the linearized plasmid in each case for a small scale expression test. Microsomes were subsequently collected and analysed by western blot (Figure 3.30). Only a very light signal is observed in the 50-75 kDa region in case of CYPntHis. This coincides with the expected MW of 60 kDa. This very low signal shows that the expression yield is low and explains why CYPnt proved so difficult to find on SDS-PAGE gel. After introduction of the Kozak sequence, no enzyme was present in the microsomal fraction, based on western blot analysis. It could thus be concluded that the introduction of this Kozak sequence did not improve the protein yield.

Figure 3.30: Western blot after small-scale expression test of three colonies picked after both cypntHis and cypntHis_KozakSeq transformation in P. pastoris. CYPntHis (60 kDa) was only produced in a low amount. No CYPntHis is present after altering the translation initiation sequence (KozakSeq).

Western blot analysis resulted only in a very low signal so other means of CYPnt identification and possibly quantification were considered. In this regard, an LC-MS method, multiple reaction monitoring (MRM), was applied for CYPnt detection and quantification in order to investigate which constructs lead to the highest enzyme yield. For MRM, there is no need for a Histag so the original CYPnt enzyme was again used. As the Kozak sequence did not lead to an increased CYPnt yield, another translation initiation sequence was investigated. The nontruncated form of CPR-a (from here on now referred to as CPRnt) shows to be produced well in P. pastoris (discussed below) and it was therefore chosen to substitute the 5’ region in cypnt with the 5’ region from cprnt, that is ACCATGGCA. Of note, this sequence also coincides with the suggested Kozak sequence as described above. This sequence was introduced using back-to-back primers in the same way as performed for the previous Kozak sequence. After transformation of the linearized plasmid in P. pastoris, a 100 ml expression test was performed, taking along the original CYPnt construct. Microsomes were isolated and prepared for MRM. In the sample preparation, BSA was added as an internal standard and the AUC was normalized using this internal standard. The AUC of CYPnt_TrlInCPRnt was adjusted relative to CYPnt and the results are shown in Figure 3.31. From this it was concluded that again, modification of the translation initiation sequence led to a decreased instead of an increased yield.

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Figure 3.31: Relative abundance of CYPnt before and after introducing the translation initiation sequence of CPRnt, determined by MRM. The total AUC for each peptide was calculated and normalized to BSA. The normalized AUC for each CYPnt peptide taken along in de analysis is shown. The normalized AUC of CYPnt was corrected to 1 and the construct with the alternative translation initiation sequence was corrected relative to CYPnt.

The alternative translation initiation sequence leads to a decreased instead of an increased yield.

3.4.2 Providing the redox partner for the nontruncated CYP52A13