Refolding of solubilised inclusion bodies

In the form of inclusion bodies the target protein is insoluble, misfolded and inactive; thus, it is necessary to refold the protein to regain its activity. Active protein can be obtained from inclusion bodies by solubilising the aggregates in high concentrations of denaturants such as guanidine hydrochloride (GdnHCl; 4–7 M) or urea (5–10 M). After the dissolution, the misfolded polypeptide can refold into its native conformation when the denaturant is removed (Sambrook and Russel, 2001). It has to be noted, however, that this approach is highly empirical and laborious.

For these experiments, three P. torridus genes were used: ORFs 615 and 810, both annotated as β-glucosidase, and 596 coding for α-amylase. The genes were initially amplified by PCR using modified primers (see section B.2.4., Table 4) and genomic DNA as a template, cloned in pCR4-TOPO and further subcloned in the expression vector pET24c using NdeI and NotI restriction sites giving p24c-615, p24c-810 and p24c-596. The expression constructs were introduced in E. coli Rosetta and expression was induced by the addition of 0.1 mM IPTG to the growth medium. SDS-PAGE analysis of the different cell fractions (soluble and insoluble, see section B.1.4.) displayed abundant inclusion body formation in all three cases which is evident from the appearance of large amounts of recombinant protein in the insoluble fraction of crude extracts (Figure 12). Also, the soluble fractions displayed no enzyme activity in assays for β-galactosidase and α-amylase respectively.

Fig. 12. SDS PAGE gel showing protein bands from inclusion bodies.(IF- insoluble fraction, R – Rosetta)

Lane 1: MWM

Lane 2: E. coliR pET24c IF Lane 3: E. coliR p24-615 IF Lane 4: E. coliR p24-810 IF Lane 5: E. coliR p24-596 IF 116

97.4 66 45

29

[kDa] [kDa]

56.252.8 44.9

1 2 3 4 5

In all the refolding methods applied, the inclusion bodies were purified (see section B.3.4.) and solubilized in 6 M guanidine HCl. An important indication of successful refolding in these cases would be the gaining of enzyme activity of the samples. Therefore enzyme activity measurements were performed with the protein samples. Another option for checking the state of the protein is to apply it to native SDS after denaturant removal. Only folded polypeptides would be able to enter the gel – high molecular weight aggregates should stay in the stacking gel.

2.1.1. Refolding by dialysis or rapid dilution

The critical factors that affect the outcome of these methods are the protein concentration and the composition of the refolding buffer (Vuillard et al., 1998).

Dialysis and rapid dilution were performed therefore with different protein concentrations of the inclusion body (IB) preparation (from 0.5 µg/ml to 10 µg/ml) and with different end concentrations of denaturant (0.01 – 0.2 M guanidine HCl) in the refolding buffer. Also, buffers with pH values ranging from 4 to 7 were tested (section B.3.4). In all three cases the dialysis resulted in precipitation of the protein. Moreover, the samples were negative for their corresponding activity and were not separable on a native PAGE (data not shown).

2.1.2. Refolding using the Vectrase kit

In these experiments, inclusion bodies obtained from the recombinant E. coli Rosetta clones (p24-615c and p24c-810) expressing the genes for the two probable β-galactosidases were used, designated as IB615 and IB810. The method screens for the refolding capacity of four different Vectrase detergents (1-4) and two Vectrase CDs (“stripping” agents, CD I and CD II). Thus eight different conditions are tested.

Analysis by native PAGE showed that two of the conditions led to the formation of soluble forms of the proteins and these conditions were the same for both samples tested (Fig. 13).

272

29 45 66

[kDa] A B

1 2 3 4 5 6 7 8 9

272

29 45 66

1 2 3 4 5 6 7 8 9

Fig. 13. Silver stained native PAGE of IB obtained from E. coliR p24-615 (A) and E. coliR p24-810 (B) after refolding with the Vectrase kit. In lanes from 1 to 8 are applied the samples from the eight different conditions, as described in the kit were applied to lanes 1 to 8: lanes 1÷4, CD I; lanes 5÷8, CD II.

The combination of “stripping” agents and detergents that led to the formation of soluble proteins was Vectrase CD I and detergent 3 for both “inclusion bodies”

proteins tested. While in the case of IB615 the soluble polypeptide had the form of a single band, IB810 gave multiple bands on the native gel which is probably due to the formation multimers with different number of monomers. However, both proteins were inactive when tested for enzyme activity.

2.1.3. Refolding using size exclusion chromatography (SEC)

The use of a gel filtration material as a medium for refolding of proteins has been described (for a review of this approach see Chaudhuri, 1994) and several examples of successful refolding have been published (Batas et al., 1999, Harrowing et al., 2003). The gel medium allows refolding to occur even at high protein concentrations by preventing inter-molecular interactions and at the same time serves to separate the refolded species from the misfolded or aggregated ones and to remove the denaturant.

Figure 14 shows the behaviour of IB615 and IB810 during such a size exclusion refolding experiment. Under these conditions, high molecular weight aggregates elute at V0, which for this column is 39.7 ml.

For these experiments the samples used were the same as in the previous method described, i.e. IB615 and IB810. 5 ml of each IB preparation (see section B.3.4.), containing 6 M guanidine HCl, were applied on a Superdex 200 16/60 column equilibrated with 50 mM phosphate buffer pH 6.5. A standard sample consisting of 3 mg each of cytochrome C, egg albumin and katalase was run under the same conditions.

The total amount of protein applied was 4.5 mg for IB615 and 10 mg for IB810. The final denaturant concentration after elution was calculated to be 0.2 M.

0

Fig. 14. Size exclusion chromatography refolding of IB615 (B) and IB 810 (C). The molecular weight standard (A) consists of 3 mg each of cytochrome C (12.5 kDa), egg albumin (45 kDa) ,katalase (240 kDa).

After 1.2 column volumes the fractions were analyzed by native PAGE (not shown) and checked for enzyme activity. The analyzed fractions showed no activity with the substrates para-nitrophenyl β D-galactoside, para-nitrophenyl α D-glucoside or para-nitrophenyl β D-glucoside . Although the peaks of the IB samples indicated soluble form of the protein, i.e. eluting after the V0 of the column, the lack of enzyme activity suggested that these forms were not the biologically native ones.