Rapid Translation System RTS

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Automated generation of Linear Templates and Biacore analysis of proteins expressed in RTS

Robot-assisted OLE-PCR of Mini BP4 Constructs


55 oligonucleotides were used to generate 31 different mini-BP4 genes by overlapping extension ligation (OLE). Site specific mutations were generated by one or two substituted and mutated primers.

Generation of Mini BP4 Linear Templates


The different gene constructs (mini-BP4 mutants) were elongated by adding a promotor and terminator module, leading to linear templates (LTs). These LTs contain the T7-promotor, g10 enhancer, ribosome binding sites, tags, cleavage sites and the T7-terminator, and can be transcribed and translated in RTS 100 E.coli HY.

32 Mutated Mini-BP4 LTs



32 different mini-BP 4 constructs were synthesized by OLE, using the RTS AviTag E.coli Biotinylation Kit, Linear Template. The PCR products are shown on an ethidium bromide stained 2% agarose gel. The first two lanes (after the marker) show intermediate constructs of mini-BP4: first a mutated mini-BP4 gene, second the same construct bridged by overlapping primers. All sequences were confirmed by sequence analysis of the linear constructs

Specific Detection of Mono-Biotinylated Mini-BP4


Non-denaturing slot blot and denaturing Western blot analyses of mini-BP4 constructs. In the slot blot (A1-G4) and Western blot (lanes 3-8), (his)₆ -mini BP4-AviTag muteins were detected via an SA-POD conjugate. The positive control in C5 / lane 1 is monobiotinylated PEX2 protein. A5 / lane 2, respectively are negative controls of RTS 100 E.coli HY reactions without template DNA. Biotin Carboxyl Carrier Protein gave no background signal in non-denaturing slot-blot (A5), but was detected after denaturing Western blot analysis. The arrows indicate corresponding samples that were detected by both methods.

Robot Performed Protein Refolding of Mini-BP4

	A: RedOx 50 mM Tris pH8 200 mM Arginine 1 mM GSH 1 mM GSSG Rmax: 102 RU Ligand activity: 20 % K_d(nM): 20 Load: 639RU	B: Oxidation 50 mM NaH₂PO₄ pH 8.0 300 mM NaCl 20 mM Imidazole 0.05% Tween 20µM CuSO₂ Rmax: 23 RU Ligand activity: 3 % K_d(nM): 20 Load: 1075RU	C: Reduction 10 mM DTT 1 mM EDTA pH 8.0 in degassed BIA HBS-EP Rmax: 0 RU Ligand activity: 0% K_d(nM): -- Load 1095RU

Interaction of mini-BP4 mutein V49F with IGF1 after robot mediated refolding of the mutein. Three different refolding conditions are shown in coloured boxes. The best condition (red box) led to mini-BP4 ligand activity of up to 20 %. Biotinylated, refolded mini-BP4 mutant V49F was immobilized on an SA-coated biosensor chip. Its interaction with IGF1 was analyzed by SPR-analysis using Biacore 3000.


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