Figure 4

Expression of candidate proteins in Escherichia coli and analysis by circular dichroism. (A) Proteins were expressed as thioredoxin (Trx) fusions in E. coli. One of the candidates shown here, R10-14, was expressed 80% or more in the soluble fraction at an expression level of ~100 mg/l. The arrow indicates the R10-14 fusion protein. -Ind denotes uninduced culture, Lys denotes lysate that contains the total protein after cell lysis, Sol is the soluble fraction after cell lysis and separation of the pellet and Insol is the fraction which contained the solubilized pellet. (B) Expression of proteins without fusion. Candidates were cloned in pCOLDII vector and expressed in E. coli after cold shock (4°C) and growth at low temperature (15°C). The expression levels were in the range 10-40 mg/l, and most of the proteins were found in the insoluble fraction. The insoluble fraction was processed to obtain inclusion bodies and purified on a Ni-NTA affinity column. The expressed and purified protein is shown as IB, denoting inclusion body, F.T denotes flowthrough, and E1 and E2 are eluates 1 and 2, respectively. R-1 μg denotes the reference lysozyme used at 1 μg. (C) Circular dichroism spectra of the refolded candidate proteins. Candidate proteins R10-13 and 14 were refolded by two methods. In the first, the reduced proteins were refolded in the presence of reduced and oxidized glutathione by dialysis as shown in (i) and in the second, the proteins were refolded in the presence of the redox system (glutathione) and immobilized protein disulfide isomerase (ii). Both spectra are from R10-14.