5 Appendix

To express and purify recombinant NE we initially used the E.coli Rosetta™(DE3)pLys strain. It is a derivative of the BL21 E. coli strain that is widely used for protein expression and is deficient in the Lon and OmpT proteases (Phillips, et al., 1984;Studier, et al., 1990). Furthermore, these commercially available bacteria carry the T7 RNA polymerase gene under the control of an IPTG inducible promoter. To avoid basal expression of the polymerase and thus unspecific expression of NE, the bacteria contained the pLys plasmid encoding for T7 lysoszyme, a natural inhibitor of T7 RNA polymerase (Studier, 1991;Zhang and Studier, 1997). To enhance the expression of eukaryotic proteins, these bacteria contain tRNAs for the translation of codons rarely used in E. coli but that are present in the NE gene.

In neutrophils, NE is synthesized as an inactive zymogen that requires the removal of amino acids at the N- and C-terminus for full activity. As a result, active mature NE starts with isoleucine instead of methionine. Since bacteria lack the proteases needed for the NE processing, we assumed that recombinant expression of the full-length protein in E. coli would not result in an active protein. Therefore, we expressed recombinant mature NE that carried an additional methionine as first aminoterminal residue. In order to purify the recombinant protein we added a histidine tag to C-terminus of the protein.

The cDNA encoding for human mature NE was ligated into the expression vector pET-28(a)+ under the control of a T7 promoter. E.coli Rosetta™(DE3)pLys were transformed with the pET28(a)+/NE mature construct and expression of recombinant NE was induced by addition of IPTG for various times at 30 or 37°C. Bacterial lysates were tested for the presence of the recombinant protein by immunoblot analysis using an antibody against histidine. However, expression of recombinant NE was not detected (data not shown).

A reason why the protein was not expressed could be based on the DNA composition of the NE gene. The 5’ sequence of this gene is very GC-rich (72%). The high GC content could lead to hairpin formation of the mRNA molecule or present an obstacle to the migration of the RNA-polymerase. In both cases protein translation would be prevented. Therefore five basepairs within the 5’-end of the DNA sequence encoding for the mature protein were silently exchanged to break GC stretches. The choice which basepairs should be mutated was based on the PROTEOEXPERT program from Roche [www.proteoexpert.com (Roche-Applied-Science, )], which is able to suggest basepair exchanges without altering the amino acid composition.

	Fig. 5.1: Mature NE expressed in bacteria is not active.
	(A) Expression of mature NE (C-terminal histidine tagged) after induction with IPTG for 4h at 37 °C. Aliquots of bacterial lysates were analyzed by SDS-PAGE and subsequent Coomassie staining or immunoblotting using an anti-histidine antibody. In the Coomasie stained gel the expressed protein is marked with an asterisk. As negative control, E.coli were transformed with the empty expression vector and also treated with IPTG. Lanes M: histidine ladder, 2: Rosetta+pET-28+(a), 3 and 4: Rosetta+pET-28+(a)/NE mature C-His, 5: 1 µg pNE (B) Purification of NE mature using a nickel column. Aliquots of each purification step were analyzed by SDS-PAGE and subsequent immunoblotting using an anti-histidine antibody. Lanes 1 and 2: flow through fractions, 3 and 4: washing fractions, 5 to 14: elution fractions, 15: Rosetta+pET-28+(a)/NE mature under non-inducing conditions; 16) starting material - Rosetta+pET-28+(a)/NE mature under inducing conditions. (C) Reconstituted recombinant NE mature analyzed by SDS-PAGE and subsequent Coomassie staining or immunoblotting using an anti-histidine antibody. Lanes M1: size ladder, M2: histidine ladder, 1 to 4: 10, 20, 50 and 75 µl of the reconstituted eluate. (D) NE activity units of 250 µl reconstituted eluate and 500 ng/ml pNE. The eluate and pNE were added to the assay buffer containing the NE peptide substrate. The kinetics were measured by recording the OD at 410 nm wave-length every 30 sec over 3 min. The NE units represent the change in OD/min.

This altered DNA was ligated into pET28(a)+ leading to a C-terminal histidine tag. E.coli Rosetta™(DE3)pLys were transformed with this construct and expression was induced. We observed that the introduced basepair exchanges lead to an expression of mature NE (figure 5.1a). In order to test the activity of the enzyme, we purified the recombinant NE using a nickel column (figure 5.1b). After dialysis, lyophilization and reconstitution of the eluted protein (figure 5.1c), its enzymatic activity was tested with the NE peptide substrate. However, we were unable to detect enzymatic activity of the recombinant mature NE protein when compared to that of purified NE (figure 5.1d).

We concluded that expression of active recombinant NE was not possible in bacteria. In a next step, we tried to express mature NE in cell-free systems (Rapid Translation System E.coli from Roche Applied Science and TNT® Quick Coupled Transcription/Translation System from Promega) or in yeast. However, using these approaches we were also not able to detect expression of recombinant NE (data not shown).

As mentioned in chapter 3.2.5, RBL-1 cells were stably transfected with the eleven different pcDNA3/NE mutant constructs. As for recombinant wildtype NE, several single-cell derived cell lines of each mutant were tested for their NE activity. After retesting three cell lines with high NE activity, the cell line whose lysate showed the highest NE activity per cell number was selected for subsequent analysis (figure 5.2). Since no cell line of the four NE mutants 58A-61, N98A, 216-218 and 216-224 cleaved the NE peptide substrate, several of these single-cell derived cell lines were tested for their ability to cleave the CG peptide substrate (figure 5.3). Again, the cell line whose lysate showed the highest CG activity per cell number was selected for subsequent analysis.

	Fig. 5.2: NE activity of different cell lines of the 11 NE mutants.
	NE activity of several single-cell derived cell lines of cells that carried the different pcDNA3/NE mutant constructs. Lysates were mixed with the NE peptide substrate and incubated for 30 min. The absorbance was read at 410 nm wave-length. The samples were normalized against a vector lysate that had been incubated with the substrate. As positive control, the substrate was added to wt lysate. All lysates were equivalent to 1x105 cells and were treated equally. The clones marked with an asterisk were chosen for further experiments.

	Fig. 5.3 : CG activity of NE mutants 58A-61, N98A, 216-218, and 216-224.
	(A) CG activity of several single-cell derived cell lines of cells individually transfected with pcDNA3/NE 58A-61, N98A, F215A or 216-224. Lysates were mixed with the CG peptide substrate and incubated for 30 min. The OD was read at 410 nm wave-length. The samples were normalized against a vector lysate that had been incubated with the substrate. As positive control, 1 μg pCG was added to vector lysate before the substrate (V+pCG). All lysates were equivalent to 1x105 and treated equally. The clones with the highest activity were chosen for further experiments. (B) CG kinetics of five single-cell derived cell lines of cells transfected with pcDNA3/NE 216-218. Lysates of 2,5x105 cell equivalence/ml were assayed for cleavage of the CG peptide substrate over 3min. The absorbance at 410 nm was read every 30 sec. As negative control the kinetics of V lysate was measured. 2,5x105 cell equivalence/ml V lysate spiked with 1,25 μg pCG was used as positive control. The CG units resembled the change in absorbance/minute.

5.3  Sequence alignment of NE, CG, trypsin and chymotrypsin

	Fig. 5.1: Sequence alignment
	The sequences of human NE (Sinha, et al., 1987), human CG (Salvesen, et al., 1987), bovine trypsin (Le Huerou, et al., 1990) and bovine chymotrypsin (Pjura, et al., 2000) were aligned using the software MUSCLE (Edgar, 2004). The numbering is according to the chymotrypsin numbering (Hartley B.S., 1971). The residues of the catalytic triad are shown in red and the amino acids that were mutated in NE are highlighted in the respective colors (see chapter 3, figure 3.11. The mature proteins start with isoleucine at position 16. The table was generated using CHROMA (Goodstadt and Pontig 2001).

5.4  Amino acids – abbreviations and structural formula

Amino acids can be grouped according to the chemical character of the respective side chain into non-polar, uncharged polar, acidic or basic residues. Figures 5.2-5.5 show the members of each group. The full name of the amino acids as well as the one- and three-letter abbreviation code is shown. The side chains are highlighted.

	Fig. 5.2: Amino acids with non-polar side chains.

	Fig. 5.3: Amino Acids with uncharged-polar side chains.

	Fig. 5.4: Amino Acids with acidic side chains.

	Fig. 5.5: Amino acids with basic side chains.