The recovered peptides gave rise to an overall good coverage in the protein sequences (Table  1). Some of the peptides recovered were unique to each protein (Figure  4, underlined). E.g., peptides SFVQEVYDYGYIPAM from LscBUpNA and SFVQEEYDYGYIPAM from LscB were located at the same position, namely 413–427, in the respective amino

acid sequences of these proteins but had different masses, 1,782 Da as compared #Temsirolimus clinical trial randurls[1|1|,|CHEM1|]# to 1,812 Da, indicating they were from different proteins. Similar differences were observed for the other peptide sequences shown in the Figure  4 indicating that the fusion constructs indeed led to the synthesis of novel fusion proteins or of the proteins intended despite the presence of similar upstream regions. Figure 4 Amino acid sequence alignment of LscB UpN A, LscB and LscB Up A. Fragments in bold indicate peptides recovered from MALDI-TOF analysis. The underlined fragments indicate recovered peptides which are unique to that protein. Table 1 Proteins identified by MALDI-TOF analysis NCBI accession number/gi Protein description Predicted molecular mass (Da) Significant hit MASCOT score Peptides matched Sequence coverage (%) 13936820 LscB 47,603 LscB 101 10 31 3914944 LscBUpNA 47,621 LscA 110 12 33 416026576 LscBUpA 45,844 LscA 110 8 19 Analysis of lscA fusion protein expression by qRT-PCR The difference in the

amount of levan produced by LscBUpA as compared to LscBUpNA and LscB in the zymogram prompted us to check if this correlated at the RNA level. Samples were grown in Z-IETD-FMK concentration HSC medium at 18°C and harvested at OD600 of 0.5 since lsc transcription is maximum at this optical density [23]. The total RNA was extracted from the cells and the expression of lscB and lscA Up B was checked by lscB-specific primers while that of lscA, lscB UpN A and lscB Up A was checked by lscA-specific primers. The results showed that, considering the standard deviation obtained for the samples, the lscB UpN A had expression levels similar to lscB (Figure  5) further

supporting the results of the Western blot and zymogram. On the other hand lscB Up A had only 60% expression as compared to lscB. As was the trend seen in the Western blot and zymogram, lscA and lscA Up B had no expression. This indicated that even though the Ureohydrolase upstream region of lscB is sufficient to promote the expression of lsc, the expression level is enhanced by the presence 48-bp N-terminus of lscB. Figure 5 Quantitative expression of different lsc genes and constructs in dependence of lscB . lscB UpN A shows similar levels of expression as lscB while lscB Up A, which does not contain the first 48 bp of lscB ORF, has lower expression. lscA and lscA Up B were not seen to be expressed. lscA, lscB UpN A and lscB Up A were detected using lscA primers (1) while the rest using lscB primers (2). The data represent the mean relative expression of 3 replicates ± standard deviations.