Biotechnology
e incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter
The correct insert size was determined by Colony PCR. Plasmids from clones containing the correct-sized inserts were isolated and the MCS was sequenced using both a forward and a reverse primer (HT7 For and Flexi R). During cloning an extra GTT, which encodes for a valine residue in the proteins' primary structures, was inserted immediately prior to each stop codon. As a universal stop codon TAA was used, replacing other stop codons if present. This was mainly done to gain maximum flexibility during cloning as the Flexi vector system enables the direct transfer to other vectors with different tags. A GTT is mandatory to later transfer the gene of interest to a vector encoding a C-terminal tag. he correct expression of the encoded fusion proteins was assessed by SDS-Page. Analyzing the gel under fluorescent conditions reveals protein bands which have the HaloTag® ligand attached. The PageRuler Plus prestained Protein ladder possesses two fluorescent bands, at 25 and 70 kDa respectively. HaloTag® Standard Protein with a size of 60 kDa was also analyzed and helps as an additional size reference. The HaloTag® features a size of 34 kDa alone.
Agarose gel of PCR products after amplification of Campylobacter jejuni genes from genomic DNA. The band sizes match the respective length of each gene. Refer to Table 2 for expected gene lengths. As markers Hyper Ladder I (M) and II (M2) were used both most of the fusion proteins investigated fall into a range between 61 and 73 kDa, namely HaloTag® fused to argC (73 kDa), pyrC (72 kDa), pseB (71 kDa), gapA (70 kDa), cjaA (65 kDa), peb1 (62 kDa), hisJ (62 kDa) and flaC (61 kDa). Outside of
The correct insert size was determined by Colony PCR. Plasmids from clones containing the correct-sized inserts were isolated and the MCS was sequenced using both a forward and a reverse primer (HT7 For and Flexi R). During cloning an extra GTT, which encodes for a valine residue in the proteins' primary structures, was inserted immediately prior to each stop codon. As a universal stop codon TAA was used, replacing other stop codons if present. This was mainly done to gain maximum flexibility during cloning as the Flexi vector system enables the direct transfer to other vectors with different tags. A GTT is mandatory to later transfer the gene of interest to a vector encoding a C-terminal tag. he correct expression of the encoded fusion proteins was assessed by SDS-Page. Analyzing the gel under fluorescent conditions reveals protein bands which have the HaloTag® ligand attached. The PageRuler Plus prestained Protein ladder possesses two fluorescent bands, at 25 and 70 kDa respectively. HaloTag® Standard Protein with a size of 60 kDa was also analyzed and helps as an additional size reference. The HaloTag® features a size of 34 kDa alone.
Agarose gel of PCR products after amplification of Campylobacter jejuni genes from genomic DNA. The band sizes match the respective length of each gene. Refer to Table 2 for expected gene lengths. As markers Hyper Ladder I (M) and II (M2) were used both most of the fusion proteins investigated fall into a range between 61 and 73 kDa, namely HaloTag® fused to argC (73 kDa), pyrC (72 kDa), pseB (71 kDa), gapA (70 kDa), cjaA (65 kDa), peb1 (62 kDa), hisJ (62 kDa) and flaC (61 kDa). Outside of