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Biomedical subjects

B Batas

Publications and source records attributed to B Batas.

4 recordsLinked to original sources
PubMed1999

Considerations of sample application and elution during size-exclusion chromatography-based protein refolding.

A mechanism for size-exclusion chromatography-based protein refolding is described. The model considers the steps of loading the denatured protein onto a gel filtration column, and protein elution. The model predictions are compared with results of refolding lysozyme (10 and 20 mg/ml) using Superdex 75 HR. The main collapse in protein structure occurred immediately after loading, where the partition coefficient of unfolded lysozyme increased from 0.1 to 0.48 for the partially folded molecule. Use of a refolding buffer as the mobile phase resulted in complete refolding of lysozyme; this eluted at an elution volume of 15.6 ml with a final partition coefficient of 0.54. The model predicted the elution volume of refolded lysozyme at 19.3 ml.

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PubMed1999

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography.

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.

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PubMed1997

Studies of the hydrodynamic volume changes that occur during refolding of lysozyme using size-exclusion chromatography.

A size-exclusion chromatography-based refolding process (SEPROS) has successfully been used to renature lysozyme at high concentrations. This process is based on the different hydrodynamic characteristics of folded and unfolded proteins and their interaction with gel filtration media. In this paper we have quantified the changes in Stokes radius, hydrodynamic volume and partition coefficient that occur when lysozyme is refolded from urea in a size-exclusion column. In 8 M urea partially folded and unfolded lysozyme were resolved using Superdex 75 HR. These two species were present at approximately the same concentration. As the urea concentration was decreased the unfolded species gradually decreased until at 4 M urea only partially folded lysozyme remained, which continued to fold on further reduction of the urea concentration. Using these results the initial mechanism for size exclusion chromatography protein refolding has been confirmed.

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