iFOLD™
`Protein Refolding Systems
`
`KASHIV EXHIBIT 1058
`IPR2019-00797
`
`Page 1
`
`

`

`iFOLD™ Protein Refolding Systems at a Glance
`
`Protein functional and structural studies often require a large
`amount of pure, correctly folded protein, which is commonly
`produced in Escherichia coli (E. coli) expression systems. How-
`ever, production of foreign proteins in E. coli can result in the
`formation of inclusion bodies (IB) — dense, insoluble, aggregates
`of misfolded protein. IB also have useful attributes — they are
`easily purified, resistant to proteolysis, and can be solubilized
`with chaotropic agents. Defi ning conditions that promote refold-
`ing of a chemically denatured protein into its native conforma-
`tion is empirical and often time consuming. Simultaneous and
`systematic evaluation of a large number of refolding conditions
`increases chances of identifying an optimal refolding condition
`for a given protein.
`
`We have taken the tedium and guesswork out of finding the
`optimal refolding condition for your target protein! Our new
`iFOLD™ Protein Refolding Systems provide comprehensive
`refolding screens in a 96-well plate format and are based on an
`extensive literature review of successful refolding experiments
`and information contained in the REFOLD database (http://refold.
`med.monash.edu.au). The systems differ in the chemistry used
`to denature the inclusion bodies and in the refolding additives
`included in the 96-well plate. In addition to a 96-well plate con-
`taining 92 (System 1) or 95 (System 2) unique buffer and refold-
`ing additive combinations, both systems include the reagents
`needed to purify inclusion bodies and solubilize the component
`proteins. Significantly, all steps of the refolding screens are
`equally compatible with manual and high-throughput automated
`liquid handling.
`
`Comparison of components in iFOLD Protein
`Refolding Systems 1 and 2
`iFOLD™ System 1
`
`iFOLD™ System 2
`
`IB wash agent
`
`IB denaturant
`
`1.0% Triton X-100
`
`0.125 M NDSB-201
`
`4.4% N-lauroylsarcosine
`
`7.0 M GuHCl
`
`IB reducing agent
`
`5 mM TCEP
`
`10 mM TCEP
`
`Denatured target protein:
`required concentration
`required volume
`
`Total Protein per well
`
`1 mg/ml
`5 ml
`50 mg
`
`Buffer system (50 mM)
`
`Tris-HCL, pH 7.0, 7.5, 8.0
`& 8.5
`
`5 mg/ml
`1 ml
`50 mg
`MOPS, pH 7.0
`HEPES, pH 7.5
`EPPS, pH 8.0
`TAPS, pH 8.5
`CHES, pH 9.0
`
`100 mM NaCl
`
`250 mM NaCl
`
`1.0 mM TCEP
`
`3.8 mM GSH + 1.2 mM
`GSSG
`
`24 mM NaCl + 1.0 mM KCl
`
`240 mM NaCl + 10 mM KCl
`
`1.0 mM TCEP
`
`9.0 mM GSH + 1.0 mM GSSG
`
`.0 mM GSH + 4.0 mM GSSG
`
`1.0 mM EDTA
`1.0 mM each of CaCl2 and
`MgCl2
`0.5 M GuHCl
`
`1.0 mM EDTA
`0.25 mM each of CaCl2, MgCl2,
`MnCl2 and ZnCl2
`0.5 or 1.0 M NDSB-201
`
`20% glycerol
`
`0.1% PEG000
`
`0.5 or 1.0 M NDSB-25
`
`1.5 M sorbitol
`
`0.58 M trehalose
`
`0.0% PEG3350
`
`Refolding additives
`
`0.5 M L-arginine
`0.5 M L-arginine
`12.5 mM methyl-β-D-
`10 mM methyl-β-D-
`cyclodextrin
`cyclodextrin
`Buffer and additive concentrations are final, after addition of the target protein
`Abbreviations: TCEP: tris(2-carboxyethyl)phosphine, GSH: reduced glutathione, GSSG: oxidized glutathione,
`EDTA: ethylenediaminetetraacetic acid, NDSB: non-detergent sulfobetaine, Sorbitol: D-sorbitol or D-glu-
`citol, Trehalose: 1-O-α-D-Glucopyranosyl-α-D-glucopyranoside, PEG000: polyethyleneglycol, average
`molecular weight of 000 Da, PEG3350: polyethyleneglycol, average molecular weight of 3350 Da
`
`Prices and availability are subject to change. ©Copyright 2007 EMD Chemicals, an affiliate of Merck KGaA,
`Darmstadt, Germany. All rights reserved. Each product is sold with a limited warranty which is provided with each
`purchase. Each product is intended to be used for research purposes only. It is not to be used for drug or diagnostic
`purposes nor is it intended for human use. EMD Chemicals products may not be resold, modified for resale, or used
`to manufacture commercial products without written approval of EMD Chemicals. NOVAGEN® is a registered
`trademark of EMD Biosciences in Australia, the European Union, Japan, Switzerland, and the United States. D-Tube™,
`D-Tube9™, IB-Prep™, iFOLD™, Lysonase™ and Perfect Protein™ are trademarks of EMD Biosciences. BODIPY® is
`a registered trademark of Molecular Probes, Inc., Brij® is a registered trademark of ICI Americas Inc., Triton® is a
`registered trademark of Dow Chemical Company.
`
`2
`
`For more information or to place an order,
`contact your local office (see back cover).
`
`Novagen • iFold Systems
`
`Page 2
`
`

`

`iFOLD™ Protein Refolding Systems
`
`Brief Protocol
`
`Express target protein
`
`Harvest cells
`
`Lyse cells by sonication
`plus Lysonase™ Bioprocessing Agent
`
`Centrifuge to pellet inclusion bodies
`
`Wash inclusion bodies with
`Triton® X-100 (System 1) or
`NDSB-201 (System 2)
`
`Denature inclusion bodies with
`N-lauroylsarcosine (System 1)
`or guanidine-HCl or urea (System 2)
`
`Refold target protein by
`rapid dilution into
`iFOLD™ Protein Refolding System 1
`or System 2 buffer matrix
`
`Assay* for correctly folded and
`active target protein
`
`*Assay is determined by end user
`
`iFOLD™ Protein Refolding System 1
`
`• All reagents for inclusion body purifi cation and pre-dispensed 96-well
` plate-based protein refolding matrix
`
`• Uses N-lauroylsarcosine, a chaotropic anionic detergent, to denature
`the purifi ed inclusion bodies
`
`• 92 unique buffer and refolding additive combinations for simultane-
`ous and systematic evaluation of protein refolding conditions
`
`• pH range 7.0 – 8.5
`
`• Refolding additives include salts, cyclodextrin, redox agents, chao-
`tropes, glycols
`
`• Refolding conditions based on extensive literature review and REFOLD
`database (http://refold.med.monash.edu.au)
`
`• High-throughput compatible
`
`iFOLD™ Protein Refolding System 2
`
`• Entirely detergent-free system 2 contains inclusion body purifi ca-
`tion reagents and pre-dispensed 96-well plate-based protein refolding
`matrix
`
`• Uses guanidine hydrochloride or urea (not included) to denature the
`purifi ed inclusion bodies
`
`• 95 unique buffer and refolding additive combinations for simultane-
`ous and systematic evaluation of protein refolding conditions
`
`• pH range 7.0 – 9.0
`
`• Refolding additives include salts, redox agents, cyclodextrin, chao-
`tropes, glycols, nondetergent sulfobetains (NDSBs)
`
`• Refolding conditions based on extensive literature review and REFOLD
`database (http://refold.med.monash.edu.au)
`
`• High-throughput compatible
`
`(cid:21) (cid:69) (cid:103) (cid:100) (cid:105) (cid:90) (cid:94) (cid:99) (cid:21) (cid:71) (cid:90) (cid:91) (cid:100) (cid:97) (cid:89) (cid:94) (cid:99) (cid:92) (cid:21) (cid:72) (cid:110) (cid:104) (cid:105) (cid:90) (cid:98) (cid:21) (cid:39)
`(cid:94) (cid:59) (cid:68) (cid:65) (cid:57) (cid:158) (cid:21) (cid:69) (cid:103) (cid:100) (cid:105) (cid:90) (cid:94) (cid:99) (cid:21) (cid:71) (cid:90) (cid:91) (cid:100) (cid:97) (cid:89) (cid:94) (cid:99) (cid:92) (cid:21) (cid:72) (cid:110) (cid:104) (cid:105) (cid:90) (cid:98) (cid:21)
`
`(cid:40) (cid:21)
`
`(cid:41) (cid:21)
`
`(cid:39) (cid:21)
`
`(cid:21)
`
`(cid:38) (cid:21)
`
`(cid:54)
`
`M O P S
`c y c l o d e x t r i n
`
`
`E D TAE D TAE D T
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`M O P S
`L - A r g
`
`E D TAE D TA
`E D T
`G S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`M O P S
`N D S B 2 5 6 ( 1 )
`m e t a l s
`
`TC E PTC E P
`T
`N a C l + K C l ( 2 )
`
`H E P E S
`t r e h a l o s e
`E D T A
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`H E P E S
`G S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`(cid:56) (cid:86) (cid:105) (cid:35) (cid:21) (cid:67) (cid:100) (cid:35) (cid:21) (cid:44)(cid:38) (cid:44)(cid:38) (cid:46) (cid:34) (cid:40)
`
`(cid:43) (cid:21)
`
`(cid:44) (cid:21)
`
`(cid:42) (cid:21)
`
`(cid:108) (cid:108) (cid:108)(cid:35) (cid:99) (cid:100) (cid:107) (cid:86) (cid:92) (cid:90) (cid:99) (cid:35) (cid:88) (cid:100) (cid:98) (cid:36) (cid:94) (cid:91) (cid:100) (cid:97) (cid:89) (cid:104) (cid:110) (cid:104) (cid:105) (cid:90) (cid:98) (cid:39)
`
`(cid:38) (cid:37) (cid:21)
`
`(cid:38)(cid:38) (cid:21)
`
`(cid:46) (cid:21)
`
`T A P S
`s o r b i t o l
`G S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`T AT A P SP S
`P E G 3 3 5 0
`E D T A
`G S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`C H E S
`s o r b i t o l
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`C H E SC H E S
`
`N D S B 2 5 6 ( 2 )
`E D T A
`G S H + G S S G ( 1 )
`N a C l + K C l ( 2 )
`
`T A P S
`G S H + G S S G ( 1 )
`
`T A P S
`N D S B 2 0 1 ( 2 )
`E D T A
`G S H + G S S G ( 1 )
`
`(cid:38) (cid:39)
`
`C H E S
`s o r b i t o l
`E D T A
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`C H E SC H E S
`
`L - A r g
`E D T A
`G S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`(cid:45) (cid:21)
`
`E P P S
` c y c l o d e x t r i n
`T C E P
`N a C l + K C l ( 2 )
`
`E P P S
`m e t a l s
`T C E P
`N a C l + K C l ( 1 )
`
`E P P S
`N D S B 2 5 6 ( 1 )
`G S H + G S S G ( 1 )
`
`E P P S
`N D S B 2 0 1 ( 1 )
`E D T A
`G S H + G S S G ( 2 )
`
`
`
`
`
`Novagen • iFold SystemsNovagen • iFold SystemsNovagen • iFold Systems
`
`(cid:56) (cid:100) (cid:98) (cid:101) (cid:100) (cid:99) (cid:90) (cid:99) (cid:105) (cid:104) (cid:21) (cid:100) (cid:91) (cid:21)
`(cid:90) (cid:86) (cid:88) (cid:93) (cid:21) (cid:108) (cid:90) (cid:97) (cid:97) (cid:21) (cid:86) (cid:103) (cid:90) (cid:21)
`(cid:104) (cid:93) (cid:100) (cid:108) (cid:99) (cid:48) (cid:21) (cid:105) (cid:93) (cid:90) (cid:21) (cid:101) (cid:61) (cid:21) (cid:94) (cid:104) (cid:21)
`(cid:94) (cid:99) (cid:89) (cid:94) (cid:88) (cid:86) (cid:105) (cid:90) (cid:89) (cid:21) (cid:87) (cid:90) (cid:97) (cid:100) (cid:108)(cid:35) (cid:21)
`(cid:56) (cid:100) (cid:99) (cid:88) (cid:90) (cid:99) (cid:105) (cid:103) (cid:86) (cid:105) (cid:94) (cid:100) (cid:99) (cid:104) (cid:21) (cid:100) (cid:91) (cid:21)
`(cid:100) (cid:105) (cid:93) (cid:90) (cid:103) (cid:21) (cid:88) (cid:100) (cid:98) (cid:101) (cid:100) (cid:99) (cid:90) (cid:99) (cid:105) (cid:104) (cid:21)
`(cid:88) (cid:86) (cid:99) (cid:21) (cid:87) (cid:90) (cid:21) (cid:91) (cid:100) (cid:106) (cid:99) (cid:89) (cid:21) (cid:94) (cid:99) (cid:21) (cid:105) (cid:93) (cid:90) (cid:21)
`(cid:105) (cid:86) (cid:87) (cid:97) (cid:90) (cid:21) (cid:100) (cid:99) (cid:21) (cid:105) (cid:93) (cid:90) (cid:21) (cid:103) (cid:90) (cid:107) (cid:90) (cid:103) (cid:104) (cid:90) (cid:21)
`(cid:104) (cid:94) (cid:89) (cid:90) (cid:21) (cid:100) (cid:91) (cid:21) (cid:105) (cid:93) (cid:94) (cid:104) (cid:21) (cid:88) (cid:86) (cid:103) (cid:89) (cid:35)
`
`(cid:66) (cid:68) (cid:69) (cid:72) (cid:33) (cid:21) (cid:101) (cid:61) (cid:21) (cid:44) (cid:35) (cid:37)
`(cid:61) (cid:58) (cid:69) (cid:58) (cid:72) (cid:33) (cid:21) (cid:101) (cid:61) (cid:21) (cid:44) (cid:35) (cid:42)
`(cid:101) (cid:61) (cid:21) (cid:45) (cid:35) (cid:37)
`(cid:58) (cid:69) (cid:69) (cid:72) (cid:33) (cid:21)
`(cid:101) (cid:61) (cid:21) (cid:45) (cid:35) (cid:42)
`
`M O P S
`L - A r g
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`(cid:55)
`
`M O P S
`P E G 3 3 5 0
`E D T A
`G S H + G S S G ( 1 )
`
`(cid:56)
`
`M O P S
`N D S B 2 5 6 ( 1 )
`
`
`E D TAE D TAE D T
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`M O P S
` c y c l o d e x t r i n
`E D T A
`
`G S H + G S S GG S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`M O P S
`N D S B 2 0 1 ( 2 )
`m e t a l s
`T C E P
`
`M O P S
`N D S B 2 5 6 ( 2 )
`m e t a l s
`T C E P
`N a C l + K C l ( 1 )
`
`M O P S
`E D T A
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`(cid:57)
`
`M O P S
`N D S B 2 0 1 ( 1 )
`
`G S H + G S S GG S H + G S S G ( 2 )
`
`M O P S
`t r e h a l o s e
`G S H + G S S G ( 1 )
`
`(cid:58)
`
`M O P S
`N D S B 2 0 1 ( 2 )
`
`G S H + G S S GG S H + G S S G ( 2 )
`
`H E P E S
`s o r b i t o l
`T C E P
`N a C l + K C l ( 1 )
`
`H E P E S
`N D S B 2 5 6 ( 2 )
`G S H + G S S G ( 1 )
`
`H E P E S
`N D S B 2 0 1 ( 1 )
`m e t a l s
`T C E P
`N a C l + K C l ( 1 )
`
`H E P E S
`L - A r g
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`H E P E S
`N D S B 2 0 1 ( 1 )
`G S H + G S S G ( 1 )
`N a C l + K C l ( 2 )
`
`H E P E S
`E D T A
`G S H + G S S G ( 2 )
`
`H E P E S
`N D S B 2 5 6 ( 2 )
`E D T A
`G S H + G S S G ( 1 )
`N a C l + K C l ( 2 )
`
`H E P E S
`N D S B 2 5 6 ( 1 )
`E D T A
`G S H + G S S G ( 2 )
`
`H E P E S
`P E G 3 3 5 0
`G S H + G S S G ( 2 )
`
`E P P S
`s o r b i t o l
`E D T A
`G S H + G S S G ( 1 )
`
`E P P S
`N D S B 2 5 6 ( 2 )
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`E P P S
`c y c l o d e x t r i n
`G S H + G S S G ( 1 )
`N a C l + K C l ( 2 )
`
`E P P S
`t r e h a l o s e
`E D T A
`G S H + G S S G ( 2 )
`
`E P P S
`N D S B 2 5 6 ( 1 )
`E D T A
`G S H + G S S G ( 1 )
`
`E P PE P P SS
` N D S B 2 0 1 ( 2 )
`E D T A
`G S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`T A P S
`N D S B 2 0 1 ( 2 )
`G S H + G S S G ( 1 )
`N a C l + K C l ( 2 )
`
`T A P S
`N D S B 2 5 6 ( 2 )
`E D T A
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`T A P S
`L - A r g
`E D T A
`G S H + G S S G ( 1 )
`N a C l + K C l ( 2 )
`
`T A P S
`N D S B 2 5 6 ( 1 )
`G S H + G S S G ( 2 )
`N a C l + K C l ( 2 )
`
`T A P S
`L - A r g
`m e t a l s
`T C E P
`N a C l + K C l ( 1 )
`
`T A P S
`t r e h a l o s e
`T C E P
`
`C H E S
`N D S B 2 0 1 ( 1 )
`E D T A
`G S H + G S S G ( 1 )
`N a C l + K C l ( 2 )
`
`C H E S
` c y c l o d e x t r i n
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`C H E S
`N D S B 2 5 6 ( 1 )
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`C H E S
`c y c l o d e x t r i n
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`E P P S
`P E G 3 3 5 0
`G S H + G S S G ( 1 )
`N a C l + K C l ( 1 )
`
`E P P S
`t r e h a l o s e
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`H E P E S
`L - A r g
`
`E P P S
`
`E P PE P P SS
`s o r b i t o l
`m e t a l s
`T C E P
`
`T A P S
` c y c l o d e x t r i n
`m e t a l s
`T C E P
`
`T A P S
`s o r b i t o l
`T C E P
`N a C l + K C l ( 1 )
`
`T A P S
` c y c l o d e x t r i n
`m e t a l s
`N a C l + K C l ( 2 )
`
`T A P S
`N D S B 2 0 1 ( 1 )
`G S H + G S S G ( 2 )
`
`C H E S
`N D S B 2 5 6 ( 1 )
`
`3
`
`T A P S
`t r e h a l o s e
`m e t a l s
`N a C l + K C l ( 1 )
`
`C H E S
`s o r b i t o l
`N a C l + K C l ( 1 )
`
`C H E S
`N D S B 2 0 1 ( 1 )
`m e t a l s
`T C E P
`N a C l + K C l ( 1 )
`
`C H E S
`P E G 3 3 5 0
`G S H + G S S G ( 2 )
`N a C l + K C l ( 1 )
`
`C H E S
`N D S B 2 5 6 ( 2 )
`T C E P
`N a C l + K C l ( 1 )
`
`C H E S
`
`Page 3
`
`

`

`Preparation of Inclusion Bodies
`
`Typically, inclusion bodies (IB) are highly enriched for the
`target protein but contain some contaminating biomol-
`ecules (nucleic acids, phospholipids, lipopolysaccharides,
`other proteins). Usually these contaminants are reduced
`by washing the IB pellet with a detergent, such as Triton®
`X-100 or sodium deoxycholate. The iFOLD™ System 1 uses
`1.0% Triton X-100 for IB washing step.
`
`When refolded proteins are used in downstream applica-
`tions that can not tolerate even trace amounts of detergent,
`we recommend the iFOLD System 2, which uses a non-
`detergent sulfobetaine, NDSB-201, to wash the IB pellet.
`NDSB-201 is a 201 Da zwitterionic compound that does not
`form micelles and is easily removed by dialysis.
`
`Comparison of a thioredoxin-GFP fusion protein from inclusion bodies
`washed with NDSB-201 or Triton® X-100
`
`The trx-GFP fusion protein was expressed in E. coli strain
`BL21(DE3) using Overnight Express™ Instant TB medium.
`Cells were lysed by sonication and IB were processed
`according to the iFOLD 2 protocol, using 1.0% Triton X-100
`
`(lanes 2-7) or 0.125 M NDSB-201 (lanes 9-14) for the IB
`washes. Samples from each step were assayed by SDS-PAGE
`(10-20% gradient gel) and detected with Coomassie blue.
`
`
`
`1 2 3 4 5  7 8 9 10 11 12 13 14 15
`
`Lane Sample
`1.
`Perfect Protein™ Markers, 10-225 kDa
`2.
`Total cellular protein
`3.
`Soluble fraction after lysis, supplemented with 1% Triton
`X-100
`Supernatant from 1% Triton X-100 wash
`4.
`Supernatant from buffer wash #1, Triton X-100 series
`5.
`Supernatant from buffer wash #2, Triton X-100 series
`.
`1% Triton X-100 washed inclusion body pellet
`7.
`Perfect Protein Markers, 10-225 kDa
`8.
`Total cellular protein
`9.
`10. Soluble fraction after lysis, supplemented with 0.125 M
`NDSB-201
`11. Supernatant from 0.125 M NDSB-201 wash
`12. Supernatant from buffer wash #1, NDSB-201 serires
`13. Supernatant from buffer wash #2, NDSB-201 serires
`14. 0.125 M NDSB-201 washed inclusion body pellet
`15. Perfect Protein Markers, 10-225 kDa
`
`NDSB-201 comparable
`to Triton X-100
`
`HRV 3C IB were washed with NDSB-201
`or Triton X-100, using the iFOLD System
`2 protocol. Washed inclusion bodies
`were denatured with 7.0 M GuHCl and
`diluted into the iFOLD Protein Refolding
`Plate 2. After refolding for 20 h at 22°C,
`reactions were dialyzed in D-Tube9™
`Dialyzers against2 x 4.0 L buffer (20 mM
`Tris-HCl, pH 8.0, 150 mM NaCl, 5.0 mM
`DTT, and 0.03% Brij®-35) overnight at
`10°C. Enzymatic activity of the refolded
`and dialyzed proteins was quantifi ed by
`measuring cleavage of a peptide substrate
`(Glu-Ala-Leu-Phe-Gln-pNa: Bachem
`L-2050). A. Graph of HRV 3C activity for
`individual wells from the refolding screen.
`B. Data grouped by refolding buffer pH.
`
`Refolded HRV 3C protease activity from IB washed
`with NDSB-201 or Triton X-100
`
`pH 7.0
`pH 7.0
`
`pH 7.5
`pH 7.5
`
`pH 8.0
`pH 8.0
`
`pH 8.5
`pH 8.5
`
`pH 9.0
`pH 9.0
`
`0.1% Triton X-100 wash
`0.125 M NDSB-201 wash
`
`A1
`
`A2
`
`A3
`
`A4
`
`A5
`
`A6
`
`A7
`
`A8
`
`A9
`
`A10
`
`A11
`
`A12
`
`A
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`Proteolytic Activity (∆A405/min)
`
`NDSB-201
`HRV 3C Triton-X
`
`3
`
`2.5
`
`2
`
`1.5
`
`1
`
`0.5
`
`B
`
`HRV 3C activity
`
`0
`6.5
`
`7
`
`7.5
`
`8
`pH
`
`8.5
`
`9
`
`9.5
`
`4
`
`For more information or to place an order,
`contact your local office (see back cover).
`
`NDSB-201 comparable
`to Triton X-100
`
`Novagen • iFold Systems
`
`Page 4
`
`

`

`Denaturing Inclusion Bodies, System 1
`
`With the iFOLD™ Protein Refolding System 1,
`the purified inclusion bodies are denatured by
`addition of TCEP and N-lauroylsarcosine and
`
`refolded by rapid dilution into the iFOLD Protein
`Refolding Plate 1.
`
`Refolded HRV 3C protease activity from IB denatured
`with N-lauroylsarcosine
`
`wells A1-B12
`pH 7.0
`
`wells C1-D12
`pH 7.5
`
`wells E1-F12
`pH 8.0
`
`wells G1-H12
`pH 8.5
`
`Enzymatic activity of
`HRV 3C protease (22 kDa)
`refolded at 22°C was
`determined by measuring
`∆A405/min, indicating cleav-
`age of a p-nitroanilide-
`labeled peptide substrate
`(Wang 1997).
`
`4.5
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`Enzymatic Activity (∆A405 /min)
`
`Reference
`Wang, Q. et al. 1997. Anal. Biochem. 252, 238.
`
`In this refolding experiment, the iFOLD System 1
`revealed that presence of residual detergent is
`essential for refolding HRV 3C protease. None of the
`14 wells with the highest enzymatic activity contain
`cyclodextrin, which acts as a detergent trap.
`
`Denaturing Inclusion Bodies, System 2
`and refolded by rapid dilution into the iFOLD
`With the iFOLD Protein Refolding System 2,
`System 2 96-well buffer matrix.
`the purified inclusion bodies are denatured
`with guanidine-HCl or urea (not included)
`
`GFP GuHCl denature
`
`Urea
`GuHCl
`
`Comparison of GFP-Trx
`fl uorescence after refolding
`from IB denatured with
`GuHCl or urea
`
`Thioredoxin-GFP inclusion bodies were isolated
`and washed with 0.125 M NDSB-201 according
`to the iFOLD System 2 protocol, denatured
`with 7.0 M guanidine-HCl or 8.0 M urea, and
`diluted into the iFOLD System 2 refolding matrix.
`Following a 24 h incubation at 22°C, refolding
`reactions were diluted 1:4 with 50 mM Tris-Cl,
`pH 8.0, and the relative fluorescent intensity
`recorded (390 nm excitation, 510 nm emission).
`
`7
`
`7.5
`
`8
`
`8.5
`
`9
`
`9.5
`
`50000
`
`45000
`
`40000
`
`35000
`
`30000
`
`25000
`
`RFU
`
`20000
`
`15000
`
`10000
`
`5000
`
`0
`6.5
`
`Novagen • iFold Systems
`
`customer.service@merckbio.com
`technical.service@merckbio.com
`Visit our website www.merckbio.com
`
`5
`
`Page 5
`
`

`

`Protein Refolding Temperature
`Following addition of the target protein, the refolding plate
`is incubated at 4-22°C for approximately 20 h. iFOLD™
`System 2 buffers maintain a relatively constant pH between
`4 and 22°C, facilitating refolding experiments at different
`temperatures to accommodate the requirements of differ-
`
`ent target proteins. For many proteins, refolding at 22°C
`results in a greater fraction of active protein than refolding
`at lower temperatures. If the target protein isthermally
`unstable or particularly difficult to refold, refolding can be
`done at lower temperatures (4-10°C)
`
`Comparison of recombinant enterokinase (rEK) activity after refolding at 10°C and 22°C
`
`(cid:38)(cid:37)(cid:149)(cid:56)
`(cid:39)(cid:39)(cid:149)(cid:56)
`
`Previously prepared rEK IB were denatured with GuHCl according to the
`iFOLD System 2 protocol. Denatured IB were diluted into an iFOLD Protein
`Refolding Plate 2 equilibrated at 10°C or 22°C. Following a 20-h incubation,
`refolding reactions were dialyzed in D-Tube9™ Dialyzers against 2 × 4.0 L
`buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl) overnight at 10°C. Enzymatic
`activity of refolded and dialyzed reactions was quantified by measuring
`cleavage of a peptide substrate (Gly-Asp-Asp-Asp-Asp-Lys-
`β-naphthylamide; Sigma G521).
`
`(cid:54)(cid:44)
`
`(cid:55)(cid:44)
`
`(cid:55)(cid:38)(cid:38)
`
`(cid:55)(cid:38)(cid:39)
`
`(cid:56)(cid:42)
`
`(cid:56)(cid:38)(cid:37)
`
`(cid:57)(cid:44)
`
`(cid:57)(cid:46)
`
`(cid:57)(cid:38)(cid:37)
`
`(cid:58)(cid:39)
`
`(cid:58)(cid:41)
`
`(cid:58)(cid:44)
`
`rEK, a protease with 9 cysteines (8 participating in disulfide
`bonds), was refolded using iFOLD System 2 at 10°C or 22°C.
`Successful refolding was determined by a protease activity assay,
`which indicated that refolding at 10°C was significantly better
`than at 22°C. Each of the 12 refolding wells with detectable
`proteolytic activity included glutathione redox buffers.
`
`(cid:38)(cid:43)(cid:37)
`
`(cid:38)(cid:42)(cid:37)
`
`(cid:38)(cid:41)(cid:37)
`
`(cid:38)(cid:40)(cid:37)
`
`(cid:38)(cid:39)(cid:37)
`
`(cid:38)(cid:38)(cid:37)
`
`(cid:38)(cid:37)(cid:37)
`
`(cid:46)(cid:37)
`
`(cid:45)(cid:37)
`
`(cid:44)(cid:37)
`
`(cid:43)(cid:37)
`
`(cid:42)(cid:37)
`
`(cid:41)(cid:37)
`
`(cid:40)(cid:37)
`
`(cid:39)(cid:37)
`
`(cid:38)(cid:37)
`
`(cid:37)
`
`(cid:69)(cid:103)(cid:100)(cid:105)(cid:90)(cid:100)(cid:97)(cid:110)(cid:105)(cid:94)(cid:88)(cid:21)(cid:54)(cid:88)(cid:105)(cid:94)(cid:107)(cid:94)(cid:105)(cid:110)(cid:21)(cid:29)(cid:244)(cid:71)(cid:35)(cid:59)(cid:35)(cid:74)(cid:35)(cid:36)(cid:98)(cid:94)(cid:99)(cid:30)
`
`Comparison of MMP-12 refolding at 10°C and 22°C
`
`10°C
`22°C
`
`Previously prepared MMP-12 IB were denatured with GuHCl according
`to the iFOLD System 2 protocol. Denatured IB were diluted into an iFOLD
`Protein Refolding Plate 2 equilibrated at 10°C or 22°C. Following a 20-h
`incubation, refolding reactions were dialyzed in D-Tube9™ Dialyzers
`against 2 x 4.0 L buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.0 mM
`CaCl2, 1 mM ZnCl2, and 0.03% Brij®-35) overnight at 10°C. Enzymatic
`activity of refolded and dialyzed reactions was quantified by measuring
`cleavage of a BODIPY®-labeled elastin (Invitrogen, E 1205).
`
`E2
`
`F2 H2 A3 C3 D4 G5 F6 B7 G7 C8 G8 D10 E11 F11 F12 G12
`
`In contrast to rEK, MMP-12 refolding was significantly better at
`22°C. The refolding screen also revealed that divalent metals (wells
`C3, C8, and F2) are required for successful MMP-12 refolding.
`
`130
`
`120
`
`110
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Enzymatic Activity (∆R.F.U./min)
`
`
`
`For more information or to place an order,
`contact your local office (see back cover).
`
`Novagen • iFold Systems
`
`Page 6
`
`

`

`Measuring Refolding
`Although no universal, accessible, high-throughput
`method is available to monitor protein refolding, sample
`absorbance at 340 nm (A340) provides an initial screen for
`refolding efficiency (Vincentelli 2004). Low A340 values
`indicate soluble proteins. Light scattering instruments
`can provide a more accurate measure of protein
`precipitation. However, as a soluble protein does not
`always correlate with a correctly folded and active
`protein, we recommend to use an activity assay specific
`for the target protein.
`
`Reference
`Vincentelli, R. et al. 2004. Protein Science. 13, 2782-2792.
`
`D-Tube 9™ and D-Tube Dialyzers
`D-Tube96™ Dialyzers allow convenient, high through-
`put dialysis of 96 samples simultaneously. The device
`features the advantages of the D-Tube™ Dialyzer Mini in
`a 96-tube format. D-Tube Dialyzers are easy to handle
`tubes with dual membranes providing a large surface
`area for fast, efficient dialysis. The membrane is ultra
`clean, EDTA-treated, regenerated cellulose that is
`sulfur- and heavy metal-free. After screening for
`optimal refolding conditions with the iFOLD™ Protein
`Refolding Systems, D-Tube96 Dialyzers provide
`convenient buffer exchange for the 96 protein samples
`into a physiologically relevant buffer.
`
`Individual D-Tube Dialyzers are available with molecu-
`lar weight cut-offs (MWCO) from 3.5 to 14 kDa and are
`designed with four volume capacities: Mini (10-250 ml),
`Midi (50-800 ml), Maxi (100-3000 ml), and Mega (3-20 ml).
`Each kit contains 10 D-Tubes and one floating rack.
`
`Product
`
`Cat. No.
`
`Size
`
`Price
`
`iFOLD™ Protein Refolding System 2
`
`71719-3
`
`1 system
`
`
`
`Components:
`• 30 mL
`• 100 µL
`• 0.5 mL
`• 10 mL
`• 10 mL
`
`
`• 1
`• 2
`
`10X IB•Prep™ Buffer
`Lysonase™ Bioprocessing Reagent
`1.0 M TCEP
`1.5 M NDSB-201
`iFOLD System 2 Denaturation Buffer
`(50 mM Tris-HCl, 0.2 M NaCl, 2.0 mM EDTA, 7.0 M GuHCl, pH 8.0)
`iFOLD Protein Refolding Plate 2
`Aluminum plate sealers
`
`iFOLD™ Protein Refolding System 1
`
`71552-3
`
`1 system
`
`
`
`
`
`10X IB-Prep™ Buffer
`1M TCEP
`Triton® X-100
`Lysonase™ Bioprocessing Reagent
`30% N-Lauroylsarcosine
`50X iFOLD Dialysis Buffer
`iFOLD Protein Refolding Plate 1
`Aluminum Plate Sealers
`
`Components:
`• 30 ml
`• 0.5 ml
`• 1.5 ml
`• 0.1 ml
`• 10 ml
`• 50 ml
`• 1
`• 2
`
`Features
`
`• Efficient dialysis – dual membrane surface
`• D-Tube9 Dialyzers are ideal for sample volume from
`10-250 ml per well/tube
`• D-Tube Dialyzers are ideal for sample volume from 10 ml
`to 20 ml per well/tube
`• Convenient – use single or multichannel pipet or
`robotic dispensing
`• High sample recovery, >97%
`• Protease-, RNase-, and DNase-free
`• Versatile – dialyze proteins, oligonucleotides, RNA, or DNA
`
`Product
`
`Cat. No.
`
`Size
`
`Price
`
`D-Tube™ Dialyzer Mini, MWCO -8 kDa
`
`D-Tube™ Dialyzer Mini, MWCO 12-14 kDa
`
`D-Tube™ Dialyzer Midi, MWCO 3.5 kDa
`
`D-Tube™ Dialyzer Midi, MWCO -8 kDa
`
`D-Tube™ Dialyzer Maxi, MWCO 3.5 kDa
`
`D-Tube™ Dialyzer Maxi, MWCO -8 kDa
`
`71504-3
`
`71505-3
`
`7150-3
`
`71507-3
`
`71508-3
`
`71509-3
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`
`
`
`
`
`
`
`
`
`
`
`
`D-Tube™ Dialyzer Maxi, MWCO 12-14 kDa
`
`71510-3
`
`D-Tube™ Dialyzer Mega, 10 ml, MWCO 3.5 kDa
`
`71739-3
`
`D-Tube™ Dialyzer Mega, 10 ml, MWCO -8 kDa
`
`71740-3
`
`D-Tube™ Dialyzer Mega, 10 ml,
`MWCO 12-14 kDa
`
`71741-3
`
`D-Tube™ Dialyzer Mega, 15 ml, MWCO 3.5 kDa
`
`71742-3
`
`D-Tube™ Dialyzer Mega, 15 ml, MWCO -8 kDa
`
`71743-3
`
`D-Tube™ Dialyzer Mega, 15 ml,
`MWCO 12-14 kDa
`
`71744-3
`
`D-Tube™ Dialyzer Mega, 20 ml, MWCO 3.5 kDa
`
`71745-3
`
`D-Tube™ Dialyzer Mega, 20 ml, MWCO -8 kDa
`
`7174-3
`
`D-Tube™ Dialyzer Mega, 20 ml,
`MWCO 12-14 kDa
`
`D-Tube9™ Dialyzer, MWCO –8 kDa
`
`D-Tube9™ Dialyzer, MWCO 12–14 kDa
`
`71747-3
`
`71712-3
`
`71713-3
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`1 kit
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`customer.service@merckbio.com
`technical.service@merckbio.com
`Visit our website www.merckbio.com
`
`77
`
`Page 7
`
`

`

`Argentina
`Merck Quimica Argentina S.A.I.C.
`Tel:
`+54 11 4546 8100
`
`+54 11 4546 8156
`Fax:
`+54 11 4546 8175
`E-mail: gbarros@merck.com.ar
`www.merck.com.ar
`
`Australia
`Merck Pty. Limited
`Tel:
`+61 3 9728 7600
`Fax:
`+61 3 9728 1351
`E-mail: merck@merck.com.au
`www.merck.com.au
`
`Brazil
`Merck S.A.
`Toll Free: 0800 21 9292
`Tel:
`+55 11 3346 8500
`Fax:
`+55 11 3207 5040
`E-mail: quimica@merck.com.br
`www.merck.com.br
`
`Imprint do Brasil Ltda.
`Tel:
`+55 19 3772 2900
`Fax:
`+55 19 3273 5389
`E-mail: imprint@imprint.com.br
`www.imprint-corp.com
`
`Caribbean
`Merck Centroamericana S.A.
`See information under Central America
`
`Central America
`Merck, S.A. Guatemala
`Tel:
`+50 2 2410-2300
`Fax:
`+50 2 2434-2954
`E-mail: quimicos@merck.com.gt
`www.merck.com.gt
`
`Chile
`Merck S.A.
`Tel:
`+56 2 3400 000
`Fax:
`+56 2 3400 199
`E-mail: mqch@merck.cl
`www.merck.cl
`
`China
`Merck China
`Toll Free: 800 820 8872
`Tel:
`+86 21 3222 4788
`Fax:
`+86 21 6247 9680
`E-mail: Bioteam@merck-china.com
`www.merckbio.cn
`
`Colombia
`Merck Colombia S.A.
`Tel:
` +57 1 425 4770
`Fax:
`+57 1 425 5407
`E-mail: mcsa@merck.com.co
`www.merck.com.co
`
`Ecuador
`Merck C.A.
`Tel:
`+593 2 2981677
`Fax:
`+593 2 2981644
`E-mail: sicmerck@merck.com.ec
`www.merck.com.ec
`
`Guatemala
`Merck Centroamericana S.A.
`See information under Central America
`
`Hong Kong
`Onwon Trading Limited
`Tel:
`+852 2757 7569
`Fax:
`+852 2757 7211
`E-mail: info@onwon.com.hk
`www.onwon.com.hk
`
`India
`Merck Specialities Private Limited
`Tel:
`+91 22 6660 9184
`Tel:
`+91 22 6660 9000
`Fax:
`+91 22 2495 4590
`Fax:
`+91 22 2495 0307
`E-mail: life.science@merck.co.in
`www.merck.co.in
`
`Indonesia
`Pt. Merck Tbk.
`Toll Free: 0800 140 1253
`Tel:
`+62 21 841 3889
`Fax:
`+62 21 841 5537
`E-mail: chemicals@merck.co.id
`
`Israel
`Mercury Scientific & Industrial
`Products Ltd.
`Tel:
`+972 3 9387164
`Fax:
`+972 3 9387174
`E-mail: mercury@mercury-ltd.co.il
`www.mercury-ltd.co.il
`
`Japan
`Merck Ltd.
`Tel:
`+81 0120 189 390
`Fax:
`+81 0120 189 350
`E-mail: service@merck.co.jp
`www.merck.co.jp
`
`Korea
`Merck Limited
`Tel:
`+82 2 2185 3836
`Fax:
`+82 2 2185 3870
`E-mail: service@merck.co.kr
`www.merck.co.kr
`
`Malaysia
`Merck Sdn Bhd
`Tel:
`+6 03 7882 4888
`Tel:
`+6 03 7880 0811
`Fax:
`+6 03 7880 0749
`Fax:
`+6 03 7880 0792
`E-mail: chemlab@merck-de.com.my
`www.merck.com.my
`
`New Zealand
`Merck Limited
`Toll Free: 0800 46 37 25
`Tel:
`+64 06 356 7328
`Fax:
`+64 06 356 7311
`E-mail: info@merck.co.nz
`www.merck.co.nz
`
`Mexico
`Control Técnico Y Representaciones
`Monterrey, Nuevo, León
`Tel:
`+52 81 8158 0600
`Fax:
`+52 81 8373 2891
`E-mail: ctrscientific@infosel.net.mx
`
`Mexico City
`Tel:
`+52 55 5208 5197
`
`+52 55 5208 5198
`
`+52 55 5208 8116
`Fax:
`+52 55 5203 6229
`
`Pakistan
`Merck Marker (Pvt) Ltd.
`Tel:
`+92 21 455 9210
`Fax:
`+92 21 453 5294
`E-mail: lab@merck.com.pk
`www.merck.com.pk
`
`Peru
`Merck Peruana S.A.
`Tel:
`+51 1 6187 500
`Fax:
`+51 1 4372 955
`E-mail: merck.peruana@merck.com.pe
`www.merck.com.pe
`
`Philippines
`Merck Inc.
`Tel:
`+63 2 815 4067
`Tel:
`+63 2 814 5221
`Fax:
`+63 2 815 4883
`E-mail: tish.aligada@merck.ph
`
`Singapore
`Merck Pte. Ltd.
`Customer Hotline: +65 6890 6660
`Tel:
`+65 6890 6638
`Fax:
`+65 6890 6636
`E-mail: chem@merck-de.com.sg
`www.merck.com.sg
`
`Taiwan
`Merck Ltd.
`Tel:
`+886 2 2742 2788
`Fax:
`+886 2 2742 2766
`E-mail: bioservice@merck.com.tw
`www.merckbio.com.tw
`
`Thailand
`Merck Ltd.
`Tel:
`+66 2 667 8333
`Fax:
`+66 2 667 8338
`E-mail: customercare@merck.co.th
`www.merck.co.th
`
`Venezuela
`Merck S.A.
`Tel:
`+58 21 2235 1379
`Fax:
`+58 21 2237 7632
`E-mail: mven@merck.com.ve
`www.merck.com.ve
`
`Vietnam
`Merck Representative Office
`Tel:
`+84 8 932 0187
`Fax:
`+84 8 526 0201
`E-mail: merckvnadmin@hcm.vnn.vn
`
`220023-2007APL A
`iFOLD Protein Refolding Systems
`
`Page 8
`
`