Refolding of recombinant proteins

Inclusion Body Purification & Protein Refolding

INQUIRY

Background

Recombinant protein expression in bacteria often results in the

formation of both inactive and insoluble protein that accumulates as

intracellularprotein aggregates called inclusion bodies [1]. It has

beenshown that 70-80% of recombinant proteins expressed in

E.coli are as inclusion bodies [2]. This is probably due to the

independence of the protein type in bacterial systems. In cases of

expression of eukaryotic proteins, which usually contain cysteines

that are prone to form disulfide bonds in the nativestate, the

bacterial system maynot support the appropriate pairing of disulfide

bonds in the newly-produced protein thus leads to the presence of

insoluble protein pellets [3].

Inclusion body purification & protein refolding

Inclusion bodies are not restricted to E.coli, they can also form in

yeast, mammalian, and insect cells. Inclusion bodies recovered

from cell lysates by low-speed centrifugation are heavily

contaminated with E.coli cell wall and outer membranecomponents.

Here is what we do at Profacgen to obtain native and soluble

protein form. First of all, the insoluble protein pellets must be

separated from other cellular components by homogenization,

washing and centrifugation; which is then followed by the refolding

of protein by solubilization in denaturants, such as guanidine

hydrochloride or urea [1]. Besides, certain reducing reagents are

added to reduce the polypeptide cysteines to break existing

disulfide bonds to obtain monomeric peptide chains [4].

Generally speaking, we apply selective extraction with detergents

and low concentrations of urea or guanidine chloride to solublize

the protein pellets. These basic steps can dissolve about 60% of

the pellet protein. The challenge, therefore, is not to purify the

recombinantly-derived protein, but to solubilize it and then fold it

into native and biologically active protein [5].

Profacgen possesses expertise in recovery of correctly folded

protein, which requires laborious and expensive processing of

inclusion bodies by conventional methods. We guarantee the

structural integrity and native conformation of your target protein.

Here we provide the most common procedure used in our

laboratory for your reference.

Experimental outline at Profacgen

Inclusion bodies are noncrystalline, amorphous structures; however,

there is evidence that the constituent densely packed proteins may

have native-like secondary structures [6]. The decision of whether

to work with insoluble recombinant protein or to put more effort into

generating soluble protein (e.g., try to modify the expression vector,

change the host strain and fermentation conditions or co-express

with molecular chaperones etc.) depends on the characteristics of

the protein.

Inclusion body purification & protein refolding

Protocol for purification of inclusion bodies & protein refolding at

Profacgen

Step 1. Preparation of inclusion bodies:

a. Harvest bacteria after induction.

b. Lyse bacteria by sonication in the buffer containing Tri-HCl,

NaCl, EDTA, NaN3, Triton-X100, PMSF and DTT. 50 ml aliquot

usually works well for sonication.

c. Add MgSO4 to chelate the EDTA,then add DNase and

lysozyme to the lysate and incubate at RT.

d. Centrifuge the mixture and collect inclusion bodies. Crush the

pellet and re-suspend by sonication in the lysis buffer. Repeat this

step.

e. Wash the inclusion bodies with lysis buffer without Triton-X100.

Re-suspend the pellet by sonication.

f. Eventually collect the inclusion body pellet by centrifugation.

Step 2. Dissolve the inclusion bodies:

a. Add Tris buffer (with Glycine) into pure inclusion body.

b. Disperse the pellets by sonication and dissolve the suspension

dropwise. Stir vigorously in Tris buffer containing urea.

c. Add GSSH (oxidized glutathione) and GSSG (reduced

glutathione), and stir overnight.

Step 3. Protein refolding:

Method I: Refolding by urea.

a. Refolding buffers include Tris and L-Arginine supplemented with

a concentration gradient Urea solution.

b. Set the pH.

c. Add EDTA, protease inhibitors and PMSF immediately before

use.

d. Dialyze against refolding buffer with concentration gradient urea

solution.

e. Dilute refolding buffer with water.

f. Dialyze against running buffer with PMSF.

Method II: Refolding by rapid dilution.

Add solubilized inclusion bodies dropwise into refolding buffer with

rapid stirring.

Generally, a large portion of misfolded aggregates and multimers

will crash out when the protein is refolded or concentrated. The

yield by mass of refolded protein from a pellet for most proteins is

about 2-5%, although some proteins refold more easily (about

20%).

Click here to contact us for more technical information.

References:

[1]Fischer B, Sumner I, Goodenough P. Isolation, renaturation, and

formation of disulfide bonds of eukaryotic proteins expressed in

Escherichia coli as inclusion bodies[J]. Biotechnol Bioeng, 1993,

41(1): 3-13.
[2]Yang Z, Zhang L, Zhang Y, et al. Highly efficient production of

soluble proteins from insoluble inclusion bodies by a

two-step-denaturing and refolding method[J]. PLoS One, 2011, 6(7):

e22981.

[3]Tyedmers J, Mogk A, Bukau B. Cellular strategies for controlling

protein aggregation[J]. Nat Rev Mol Cell Biol, 2010, 11(11): 777-88.

[4]O'callaghan C A, Tormo J, Willcox B E, et al. Production,

crystallization, and preliminary X-ray analysis of the human MHC

class Ib molecule HLA-E[J]. Protein Sci, 1998, 7(5): 1264-6.

[5]Palmer I, Wingfield P T. Preparation and extraction of insoluble

(inclusion-body) proteins from Escherichia coli[J]. Curr Protoc

Protein Sci, 2004, Chapter 6: Unit 6 3.

[6]Oberg K, Chrunyk B A, Wetzel R, et al. Nativelike secondary

structure in interleukin-1 beta inclusion bodies by attenuated total

reflectance FTIR[J]. Biochemistry, 1994, 33(9): 2628-34.