Materials and Methods
Choice of linkers. Nat-Quarter : N- NATPTKGAT-C (9 residues); Nat-Half : N-NATPTKGATPTNTATPTKSAT-C (21 residues);Nat-Full : N-NATPTKGATPTNTATPTKSATATPTRPSVPTNTPTNTPANTP-C (42 residues); Rigid : N- EAAAKEAAAKEAAAK-C (15 residues);Flexible : N- SGGGGSGGGG-C (10 residues). Nat-Quarter andNat-Half describe the first 9 and 21 residues, respectively, ofNat-Full , a linker lying on the C-terminal side of Coh2 inC. thermocellulm CipA. Rigid is an archetypal rigid linker, consisting of 3 repeats of N-EAAAK-C. Flexible is an archetypal flexible linker, consisting of 2 repeats of N-SGGGG-C.
Construction of fusion proteins. To link Coh2 and BSX with the above linkers, (i) DNA encoding Coh2 was PCR-amplified from C. thermocellum genomic DNA (strain ATCC 27405); and (ii) DNA encoding BSX was gotten commercially synthesised. Coh2 and BSX were fused through splicing by overlap extension (SOE) PCR, using appropriately-designed primers incorporating the said linkers. Coh2-linker-BSX-encoding fusions were PCR amplified, digested by NdeI, and XhoI, and ligated into the T7-based expression vector pET23a, to be produced in fusion with a C-terminal 6xHis affinity tag.
Protein expression and purification. Plasmid vectors (pET23a) bearing genes encoding Coh2-BSX fusions were transformed into XL-1 BlueE. coli to produce plasmids for DNA sequencing that were later transformed into E coli BL21 Star (DE3) pLysS, towards protein over-expression and purification. Overexpression was induced during growth of transformed cells in LB media by 1 mM IPTG in the mid-exponential phase of culture growth, at an optical density (O.D.600) of 0.6. Following induction, cells were incubated for 8 hours at 37 °C, sedimented and then lysed. Expressed 6xHis-tagged protein fusions were chromatographically purified from clarified lysates through Ni-NTA chromatography, using columns from GE Healthcare. Protein yields were typically 4 to 5 mg per litre of culture. All five Coh2-BSX fusion proteins were purified under non-denaturing conditions and transferred into 20 mM Na-HEPES buffer (pH 7.5, containing 100 mM NaCl, and 2 mM CaCl2) through buffer-exchange upon a Superdex-75 size exclusion chromatography (SEC) column from GE Healthcare, using the GE Akta Purifier 10 chromatographic workstation. The same set up was also used in analytical mode, to examine elution behaviour of Coh2-linker-BSX fusion proteins incorporating different linkers, following 40 days of storage.
SDS–PAGE analysis. SDS-PAGE gels (12 %) were used to examine the covalent (chain) integrity of Coh2-linker-BSX protein fusions. Protein markers with molecular weights ranging from 14.4 kDa to 116 kDa were run alongside. To detect proteolytic degradation, SDS-PAGE was performed both immediately following purification (0 days), and also following 15 days of storage at 4 °C.
Proteolysis-susceptibility assay . Subtilisin A (a ~27 kDa serine protease) from B. licheniformiswas sourced from Merck (Sigma), USA. The protease was added to the Coh2-linker-BSX fusion made using the Flexible linker (i.e., Coh2-Flexible -BSX), to examine the linker’s degree of resistance to proteolysis by exogenously-added Subtilisin A, which is known to hydrolyse peptide bonds without any sequence specificity (Markland, 1971). Coh2-Flexible- BSX was incubated with Subtilisin for 2 h at 50 °C, using Subtilisin A:Coh2-Flexible- BSX molar ratios varying from 1:1 to 1:1000 (or 0.001:1), using stock solutions of Subtilisin A prepared in 20 mM Na-HEPES buffer (pH 7.5, containing 100 mM NaCl, and 2 mM CaCl2) and stock solutions of Coh2-Flexible- BSX as mentioned above. Samples were analysed on 12 % SDS–PAGE to assess proteolytic degradation.