You are logged on as Guest
`
`Sign in
`
`Register
`
`Home
`
`About Us
`
`Products
`
`Services
`
`Facilities
`
`App & Tech
`
`Contact Us
`
`USA
`
`24x7 Custom Service
`
`INQUIRY
`
`Product Search
`
`Search
`
`Home / Inclusion body purification & protein refolding
`
`App & Tech
`Applications & Technologies
`
`Inclusion body purification & protein refolding
`
`Recombinant protein expression in E.coli
`
`Recombinant protein expression in Insect cells
`
`Recombinant protein expression in Pichia pastoris
`
`Co-Immunoprecipitation (Co-IP)
`
`Yeast Two-hybrid Screening
`
`Pull Down Assay
`
`Recombinant Protein Expression in Mammalian Cells (HEK293/CHO)
`
`CONTACT US
`
`Our promise
`to you :
`Guaranteect
`product quiality,
`eJIIPert oust:omer
`s,upport.
`
`1 of 5
`
`Fresenius Kabi
`Exhibit 1012
`
`

`

` Tel: 1-631-559-9269
`1-631-448-7888
` Fax: 1-631-938-8127
` Email: info@profacgen.com
`
`Inclusion body purification & protein refolding
`
`INQUIRY
`
`Background
`Recombinant protein expression in bacteria often results in the formation of both inactive and insoluble protein that
`[1]
`accumulates as intracellularprotein aggregates called inclusion bodies
`. It has beenshown that 70-80% of recombinant
`[2]
`proteins expressed in E.coli are as inclusion bodies
`. 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-
`[3]
`produced protein thus leads to the presence of insoluble protein pellets
`.
`r -
`-
`-
`a Bacteria
`
`- - - ·-,
`
`b Veiast
`
`Diffusion
`
`Aggregate
`
`Polyubiq urtin
`Misfolded
`
`cMammals
`
`HDAC6
`
`Dynetn
`
`•
`
`Pola r aggregate
`(inclusion b.ody)
`
`I
`%
`
`IPOD
`
`JUNQ
`
`Aggreso me
`
`In bacteria, misfolded proteins can accumulate in inclusion bodies under different
`conditions, which most often occurs during the heterogenous expression of
`proteins. Inclusion bodies often fo rm at the periphery of the cell! .. Energy-driven
`active processes may contribute to the deposition of misfolded proteins in
`indusion bodied ..
`
`L
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`·
`
`-
`
`-
`
`-
`
`-- -- -- -- ---
`
`-
`
`-
`
`·
`
`-
`
`-
`
`-
`
`-
`
`..J
`
`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
`
`2 of 5
`
`Fresenius Kabi
`Exhibit 1012
`
`

`

`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
`[5]
`to purify the recombinantly-derived protein, but to solubilize it and then fold it into native and biologically active protein
`.
`
`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
`[6]
`proteins may have native-like secondary structures
`. 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.
`
`Reloldm,g by IRapid
`Dilution
`
`Or
`
`Refoldin,g by
`Ure
`
`Disso vet , e indusaon bodies
`
`L------------- --------"'
`
`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.
`
`3 of 5
`
`Fresenius Kabi
`Exhibit 1012
`
`

`

`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.
`
`INQUIRY
`
`Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Tel: 1-631-559-9269 1-631-448-7888 Fax: 1-631-938-
`8127 Email: info@profacgen.com
`Copyright ©2010 - 2016 Profacgen. All rights reserved | Term and Conditions | Privacy Policy
`
`4 of 5
`
`Fresenius Kabi
`Exhibit 1012
`
`

`

`5 of 5
`
`Fresenius Kabi
`
`Exhibit 1012
`
`5 of 5
`
`Fresenius Kabi
`Exhibit 1012
`
`