Recombinant production of CYPtr in a HAC1 co-expressing strain

The use of the HAC1 co-expressing strain showed promise in recombinant CPRtr production in that a markedly higher yield could be obtained, based on the peak area of the 100 mM imidazole fraction containing CPRtr. Additionally, instead of two bands, only one band appeared in both SDS-PAGE and western blot analysis, indicating that the α-factor secretion signal was correctly processed only in this HAC1 co-expressing strain. After producing CYPtr in the wild type strain NRRL-Y-11430, the enzyme was not secreted and appeared at a higher MW, indicating that the secretion signal was not processed. Therefore, cyptr was transformed in this HAC1 co-expressing strain to see if also for this enzyme, the processing of the secretion signal was aided.

Three colonies were taken along in a small scale expression test. A medium sample was concentrated and analysed by western blot, together with the lysate and the isolated microsomes (Figure 3.35). Remarkably, CYPtr was found in the medium in case of the second colony. A band is seen around the expected MW of 58 kDa, whereas this is not visible in case of the first and third colony. However, the enzyme is still partially retained in the microsomal fraction.

Figure 3.35: Western blot of concentrated medium, lysate and microsome samples after small-scale expression test of three colonies picked after cyptr transformation in the HAC1 co-expressing strain. CYPtr (58 kDa, 67 kDa in case of an unprocessed signal sequence) is successfully produced and secreted in case of colony 2. In colony 1 and 3, no secreted enzyme is observed and CYPtr is largely degraded.

This observation prompted us to evaluate whether the chimeric constructs from 3.3 could be secreted as well using the HAC1 co-expressing strain. Three colonies of each chimeric construct obtained after transforming this P. pastoris strain were picked for a small-scale expression test.

A medium sample was concentrated and microsomes were isolated for western blot analysis (Figure 3.36). Unfortunately, no enzyme was observed in the medium and the chimers were still retained in the microsomes.

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Figure 3.36: Western blot of concentrated medium and microsome samples after small-scale expression test of three colonies picked after chimer transformation in HAC1 co-expressing P. pastoris. A: CYPtrCPRtr (131 kDa, 141 kDa in case of an unprocessed signal sequence), obtained by fusion PCR and by the substitution approach. B:

CYPtrBMR (123 kDa, 132 kDa in case of an unprocessed signal sequence). Both chimeric constructs were not secreted but were retained in the microsomal fraction.

We continued investigating colony 2, able to secrete CYPtr into the medium. Cells were grown in a 500 ml culture, followed by IMAC purification of the medium (Figure 3.37) and the different elution peaks were analysed by SDS-PAGE (Figure 3.38). No clear band was observed around the MW of 58 kDa, the expected MW of secreted CYPtr. In both the 10 mM and 100 mM imidazole elution peak, an intense band was observed at a lower MW. In the 100 mM imidazole elution peak, two additional bands were seen. All these gel bands were cut out for MALDI-TOF MS. Unfortunately, CYPtr could not be identified and elution peaks contained contaminants only.

Figure 3.37: IMAC chromatogram of CYPtr purification from the medium after expression in a 500 ml culture.

The right chromatogram shows the elution profile in more detail.

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Figure 3.38: Analysis of IMAC elution peaks, desalted and concentrated after CYPtr (58 kDa) purification Figure 3.37. The gel bands were cut out and analysed by MALDI-TOF MS. The higher MW gel band in the 100 mM imidazole elution peak was identified as dihydroxyacetone synthase variant 2 (DAS2).The gel band in between 37 kDa and 50 kDa, present in both the 10 mM and 100 mM imidazole elution peak were identified as formate dehydrogenase (FDH). No CYPtr was identified.

Efforts were made to improve the yield of secreted CYPtr. To this end, colony 2 was further investigated in 2 ml culture experiments using 24-well deep-well plates. Using these milli-scale cultures allowed for testing multiple different additives and constructs to be performed in triplicate. In a first experiment, it was tested whether medium supplementation with either iron, δ-aminolevulinic acid (5-ALA), hemin or a combination of all three would increase CYPtr yield in the medium. Even though P. pastoris possesses the heme biosynthetic pathway and CYP intracellular production in P. pastoris has been accomplished without medium supplementation [328]–[333], the endogenous heme might not suffice to support the recombinant production and secretion of CYPtr. For example, for the recombinant production and secretion of another heme protein, heme peroxidase, in P. pastoris, the addition of hemin increased the yield of active enzyme in the medium [334]. Seven different conditions were tested and compared to the non-supplemented condition. Each of the three additives were added separately in two different concentrations, and a combination of the three additives was used in the seventh condition. The medium was collected after growth and recombinant CYPtr production and concentrated for subsequent western blot analysis (Figure 3.39). In each case, CYPtr was successfully secreted and indeed, more intense bands were observed although not to a large extent. Especially the supplementation with 0.5 mM 5-ALA and with a combination of the three additives consistently showed more intense bands across all three blots.

Figure 3.39: Western blot of DOC-TCA concentrated medium samples after milli-scale expression test in 24-well deep-well plates. Seven different medium supplementations were tested and compared to a non-supplemented culture. The three blots show the three biological replicates. * = 0.1 mM FeSO4, 0.1 mM 5-ALA and 3 µM hemin.

101 It has been mentioned above that co-expression of CPR can markedly influence CYP production and stability. In parallel to the construction of a cypntHis-cprntHis co-expression vector, a cyptr-cprntHis co-expression vector was created. Eight colonies were picked upon transformation, of which only four contained both cyptr and cprntHis, based on colony PCR.

These four colonies were subsequently grown for a small-scale expression test after which a medium sample was taken, concentrated and analysed by western blot. It was seen that CYPtr was present in the medium in a higher yield after CPRntHis co-expression (Figure 3.40A).

CPRntHis indeed was produced, as shown by western blot of the isolated microsomes (Figure 3.40B). Of note, CYPtr contains an N-terminal Histag, whereas the Histag of CPRntHis is present at the C-terminal end. The anti-His antibody used for CPRntHis detection by western blot is specific for the Histag at the C-terminus. The signal seen in the microsomes (Figure 3.40B) is thus expected to be specific for CPRntHis and should not give a false positive due to CYPtr retained in the microsomes. The MW of CPRnt is much higher than of CYPtr (both enzymes differ in MW by 20 kDa). However, they appear to be equal in MW on blot raising questions about the nature of the signal in the microsomal extract. It must be noted that the western blot marker is not suited for exact MW determination, especially in the higher MW region, as in this region the marker is often overdeveloped and consequently, individual marker bands become more difficult to distinguish.

Figure 3.40: Western blot after small-scale expression test of four colonies picked after cyptr-cprntHis co-expressing vector transformation in HAC1 co-co-expressing P. pastoris. A: Concentrated medium samples. The anti-His-HRP antibody (MA1-21315-HRP, Invitrogen) was used for CYPtr (58 kDa) detection. B: Microsomes. The anti-His(C-term)-HRP antibody (R931-25, Invitrogen), specific for C-terminal Histags, was used for CPRntHis (78 kDa) detection.

Further efforts for CYPtr yield improvement and purification optimizations were set aside. It was decided to focus on obtaining an active enzyme first because only then it becomes of interest to also optimize the production and purification process.

A B

102

4 Discussion