Refolding human serum albumin at relatively high protein concentration

Abstract

Thc conditions for refolding reduced and denatured human serum albumin (HSA) were investigated with a view to maximising the yield of native monomeric albumin. Refolding by dialysis was found to be preferable to dilution as a means of chaotrope (urea) and reductant (2-mercaptoethanol) removal. Dialysis of denatured HSA solutions containing 4-8 M urea and 14 mM 2-mercaptoethanol at pH 10.0 was found to be optimal for HSA refolding. The yield of monomeric HSA was maximal (94%) for dialysis in the presence of EDTA (1 mM) and sodium palmitate (20 pM). Using this protocol it was possible to refold HSA at concentrations in excess of 5 mg . ml-' whilst maintaining a high recovery of native monomer. These results represent a considerable improvement on established methods of HSA refolding.A common approach to solving the enigma of protein folding has been to observe the refolding of reversibly denatured proteins in vitro. The intrinsic ability of large, disulphide-bonded proteins to refold correctly from a reduced and denatured state was demonstrated by Anfinsen and coworkers from their studies on ribonuclease [l, 21. Since then a number of proteins have been refolded to their original native conformations [3 -61. Traditional protocols for refolding reduced and denatured proteins involve the removal of the chaotrope and thiol reductant by dilution or dialysis in an oxidative environment. To combat the frequently encountered problems of protein aggregation and incorrectly formed disulphide bonds, the denatured protein is commonly diluted to micromolar concentration and refolded in the presence of disulphide/thiol containing buffers [4, 71 or disulphide exchange enzymes [8, 91. Interest in protein refolding has intensified in recent years with the advent of recombinant DNA technology. Heterologous proteins expressed in microorganisms are frcquently insoluble within the cell and their recovery in a soluble form often necessitates renaturation of a chaotrope-reductant extract. Large-scale protein refolding places high demands on the renaturation protocol, since long refolding times, large volumes of dilute protein solution and poor recoveries of native material are unacceptable in a production process. Thus a requirement exists for highly efficient protein refolding regimes.Serum albumin provides an interesting model for protein refolding studies since, in order to regenerate the native protein 17, disulphide bonds must be correctly reformed [3]. Human serum albumin (HSA) is also a valued therapeutic product which has been expressed as an intracellular recombinant protein in a number of organisms [lo-121. In the majority of these examples the recombinant HSA forms C,'itrrespontf~~nce to A. V. Quirk,

Expression, Refolding, and Isotopic Labeling of Human Serum Albumin Domains for NMR Spectroscopy

“…We investigated the refolding of the HSA domains at various pH values (4 to 10) and found that an alkaline buffer (pH 10.0 to 10.5) gives the best refolding yields for all the HSA domains. Since the thiol group of cysteine residues typically has a pK a of 8 to 10, a basic condition promotes disulfide bond formation and therefore is more favorable for the refolding (9). By the same token, acidic pH may be more effective in reducing disulfide bonds by protonating the thiol groups.…”

“…It was critical to thoroughly dialyze the proteins prior to refolding, because some of the bound nickel (a group II metal) can interfere with proper disulfide bond formation (9). The completely reduced and denatured HSA domains can be refolded by dialysis using a procedure similar to that used in the refolding of full-length HSA (9). We investigated the refolding of the HSA domains at various pH values (4 to 10) and found that an alkaline buffer (pH 10.0 to 10.5) gives the best refolding yields for all the HSA domains.…”

Effect of rate of chemical or thermal renaturation on refolding and aggregation of a simple lattice protein

“…Dialysis and diafiltration have been shown to provide better refolding yields than dilution, even at relatively high protein concentrations (Burton et al, 1989;London et al, 1974;Persson and Gekas, 1994;Yoshii et al, 2000). Both methods employ a membrane that allows the passage of small denaturant molecules but retains the large protein molecules.…”

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

“…14 Compared sequences of BSA and HSA are found to be of a striking homology (around 80%). Whilst studies which concern themselves with the unfolding/refolding of serum albumins exist, 13,16 to the best of our knowledge the existing literature does not include essential physical chemical parameters, notably the Gibbs free energy and the rate constant for their reductive unfolding. Among the experimental techniques, which are suitable for monitoring thiol/disulfide exchange reactions (reaction 1), Raman spectroscopy is particularly interesting.…”

“…1 Bovine serum albumin (BSA) and human serum albumin (HSA) provide interesting models for protein unfolding and refolding studies since they contain as many as 17 disulfide bridges each. 13,14 Albumins are the most abundant proteins in blood plasma and also act as the transport protein for numerous endogenous and exogenous compounds. 15 The secondary structure of these proteins is for the most part helical (70%) however, a detailed tertiary structure as determined by X-ray is only known for HSA.…”