Cloning and expression of frs genes

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phylogenetic neighbours. Both have the active site triad of serine, aspartic acid and histidine, which is common for most TE domains.⁴⁰ There are some NRPS TEs known to have a catalytic cysteine residue instead of serine like in the polymyxin synthetase but it is quite uncommon for type I TEs.¹³⁸ In Figure 4.6 the I-TASSER-calculated structure of FrsATE (A) and an alignment of the structural models of FrsATE and FRsGTE (B) is shown. The structures of the TE domains show a repeating β/α/β motif that forms a six-stranded parallel β-sheet with a left-handed helical twist and two α-helices forming a lid over the active site with the binding serine. This general structure is similar to the structures of reported type 1 TEs.⁴⁰ The alignment of FrsATE and FrsDTE shows only some small variances, and the location of the amino acids in the active site is nearly identical.

Figure 4.6: Structural models of the TE domain of FrsA and FrsG. A The I-TASSER model of FrsATE, β-sheets are displayed in orange, α-helices are displayed in cyan and the two helices of the lid in blue. The amino acids (Ser, Asp and His) of the active site are displayed in orange. B Alignment of the structural models of FrsATE (orange) and FrsGTE (white) from C.

vaccinii.

The highest similarity for existing structures of TE domains to the structure of FrsATE calculated by I-TASSER is NocBTE (PDB: 6ojdA). The latter is a bifunctional domain that catalyses not only hydrolysis but also epimerisation in the biosynthesis of nocardicin.¹³⁹ Thus, it is a very special TE as well, but it does not clade very near to the frs TEs in the phylogenetic analyses. To verify the similarities and differences of these three domains in detail, the crystal structures of FrsATE and FrsGTE would be needed.

In summary, predicting the function of TE domains from the primary structure TE is not as facile as for other domains. They vary in function and structure and do not have high sequence similarities, even when catalysing similar reactions. To get more insights into these domains in the frs BGC, we wanted to investigate them in vitro and therefore progressed with their cloning and heterologous expression.

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both modules were cloned and heterologously expressed in E. coli to perform in vitro reconstitution assays. Additionally, the modules were expressed in truncated single, di or three domain constructs.

FrsB was coexpressed to assist in different assays and FrsH was expressed separately in cases where hydroxylation was needed. To compare the two TE domains, the TE of FrsG was also expressed in a single domain construct. All relevant constructs for this work are summarised in Table 4.2.

Table 4.2: Heterologously expressed proteins used in this work.

Protein Calculated molecular weight Coexpressed with:

FrsA 142.35 kDa FrsB

FrsACAT 116.46 kDa FrsB

FrsAAT 67.76 kDa FrsB

FrsAA 59.17 kDa FrsB

FrsATE 31.49 kDa -

FrsB 8.19 kDa -

FrsD 116.16 kDa FrsB

FrsGTE 30.98 kDa -

FrsH 63.75 kDa -

To generate new expression constructs, the gene sequences of the selected domains were amplified via PCR with specific primers (see Table 6.14) from the gDNA of C. vaccinii. By using selected restriction sites, the PCR products were ligated into the bacterial expression plasmid pET28a. These new plasmids, listed in Table 6.18, were transformed into E. coli α-Silver-Select and the correct sequence verified by Sanger sequencing. Afterwards, the plasmids were transformed into an E. coli expression strain and the expression was induced with IPTG and analysed. The proteins were all expressed with an N-terminal hexahistidine tag for Ni-NTA affinity purification. The first test expressions were used to see if the protein of the expected size is expressed in soluble form and in detectable amounts so that it can be purified by the chosen method. All fractions from the affinity chromatography were analysed via SDS-PAGE, for details, see section 6.6. In Figure 4.7, the SDS-PAGE of the first attempted expression of FrsAAT, coexpressed with FrsB in BL21, is shown as an example for the protein purification. The protein band in the elution fractions between 60 and 70 kDa fits the calculated mass of FrsAAT with 67.76 kDa and an additional band at approximately 8 kDa matches the coexpressed FrsB which is co-eluted with the A domain even though it has no His6-tag. The band of FrsAAT is not visible in the non-induced sample, which was taken from the expression culture before adding IPTG and lysed separately. This reassures that the expression is dependent on the induction of the T7-polymerase, as expected in a DE3-expression strain.¹⁴⁰ The N-terminal His6-tagged FrsAAT yielded the highest amount of tagged protein in the first elution fraction, whereas most unspecific binding proteins are eluted in the two wash steps.

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Figure 4.7: SDS-PAGE of FrsAAT test expression in BL21 coexpressed with FrsB. NI= non induced sample, I= induced sample, FT= flow through, W1= washing step 1, W2= washing step 2, E1= elution fraction 1, E2= elution fraction 2, E3=

elution fraction 3, P= pellet.

If the protein was expressed in sufficient amount and purity, the combined elution fractions were used for further investigation. Otherwise, the expression conditions and the purification protocol were optimized, or a new expression construct needed to be designed.

In Figure 4.8, SDS-PAGE gels of all proteins used for bioassays are pictured with their respective calculated molecular masses. All proteins including an A domain were coexpressed with FrsB to ensure the solubility and activity of the protein. All constructs harbouring a T domain were conducted in the expression strain BAP1, which carries the sfp phosphopantetheinyl transferase gene to ensure in vivo phosphopantetheinylation of the T domain.¹⁴¹ Interestingly, nearly all proteins migrate further than their calculated molecular weight. It has been discussed in different publications, that some proteins do not migrate at the estimated band hight due to their hydrophobicity.^142,143 Shirai et al. suggested, that SDS preferentially binds to the hydrophobic instead of the negatively charged regions of proteins, which may cause inconsistencies regarding theoretical mobility when comparing chemically diverse proteins.¹⁴² As our proteins were proven to be expressed from the particular plasmid, and to be bioactive (see below), we did not investigate this unusual mobility any further.

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Figure 4.8: SDS-PAGEs of all purified proteins used in this work for bioassays. The names of the proteins are located on top of the lanes and the calculated molecular masses of the proteins are shown above the bands.