If you give more physical parameter about protein, i can help in better way and i will give you a protocol for refolding and purification also. 

First try to purify inclusion bodies after lysis, used required concentration of triton X100 (1%) tween (0.1%) and Nacl (50-200 mM) again depend on protein.

if inclusion bodies are purify you can easly refold by pulse dilution with appropriate buffer... if i know PI than i can suggest any thing.

keep try

Surya

I do get almost clean protein after purifying IB. Always face problem in refolding.

Number of amino acids: 119

Molecular weight: 13492.2

Theoretical pI: 7.07

Amino acid composition:

Ala (A) 3 2.5%

Arg (R) 7 5.9%

Asn (N) 6 5.0%

Asp (D) 5 4.2%

Cys (C) 3 2.5%

Gln (Q) 3 2.5%

Glu (E) 5 4.2%

Gly (G) 11 9.2%

His (H) 4 3.4%

Ile (I) 3 2.5%

Leu (L) 11 9.2%

Lys (K) 3 2.5%

Met (M) 1 0.8%

Phe (F) 8 6.7%

Pro (P) 9 7.6%

Ser (S) 12 10.1%

Thr (T) 4 3.4%

Trp (W) 3 2.5%

Tyr (Y) 7 5.9%

Val (V) 11 9.2%

Pyl (O) 0 0.0%

Sec (U) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 10

Total number of positively charged residues (Arg + Lys): 10

Instability index:

The instability index (II) is computed to be 45.78

This classifies the protein as unstable.

Aliphatic index: 75.21

Grand average of hydropathicity (GRAVY): -0.225

Protein molecular wt is 13.4 Kda and PI is 7.07

Three cysteine is there it means one is not with disulfide bound, open cysteine is little difficult in process every were.

Refolding condition you can try with high pH 10.5-11.0 with 10-20 mM Glycine with 50-100 mM of Arginine, if still it is precipitating you can use Sorbitol/sucrose at 10% W/V or 0.1% Tween 20/80. 

Refolding temperature also you have to check do it in cold condition at 2-80C.

Stabilization with DTT/BME conc. (mM) of reducing agents depend on you protein concentration.

Refolding dilution: pulse dilution or do it in two time with very less flow rate means (2 - 5 ml/min), still is aggregating...

Refolding concentration (mg/ml) you have to decrees like if you are doing at 1 mg/ml decrees to 0.2 mg/ml or 0.1 mg/ml.

it will happen, try it....

I have tried to refold the protein at pH 8.5 (Tris-HCl) with salt varying from 100-500 mM with/without Arginine (100-500 mM) and with variable concentration of DTT (1-3 mM) at  protein concentration of (1 mg/ml or 0.5 mg/ml). Thanks for the suggestion, I will try with glycine pH 11.

If you have three cysteines in your protein, the odd one that's not involved in a disulphide bond could be causing trouble. If everything else fails and if you know which one it is, and of course if it doesn't play a crucial role, it might be worth trying to mutate it to serine or alanine and see if it rescues the folding. Alternatively, is expression in a mammalian system an option for you? Some proteins simply aren't amenable for bacterial expression.

Thanks Pavel,

If refolding at pH 10/11 doesn't work, then I will go for it. I know the residue which is not involved in the disulphide bond formation. The extracellular domain of the protein is 516 AA long and it contains 5 Ig-like domain. I am working on the N-terminal domain, it is a Ig-like V-type domain. The free Cys in the ROI forms disulphide bond with Cys of other domain.

Protein is still precipitating with pH 11 Glycine-NaOH, 50 mM L-Arg. will add 10% sucrose/tween 20 and try.