Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

Das, Mili; Kobayashi, Masanori; Yamada, Y.; Sreeramulu, Sridhar; Ramakrishnan, C.; Wakatsuki, Soichi; Kato, Ryuichi; Varadarajan, Raghavan

doi:10.1016/j.jmb.2007.07.033

Cited by 19 publications

(20 citation statements)

References 72 publications

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“…The free energies of unfolding at zero denaturant concentration (ΔG°) were found to be 8.9±0.1, 8.7±0.1 and 7.5±0.1 kcal/mol for WT Trx, pelB Trx and malE Trx, respectively. Similar values of unfolding free energies have been previously determined for WT Trx [52], [53]. A similar destabilization of malE MBP relative to mature MBP lacking the malE signal peptide was seen previously [49].…”

Section: Resultssupporting

confidence: 85%

Effect of Signal Peptide on Stability and Folding of Escherichia coli Thioredoxin

et al. 2013

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The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

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Section: Resultssupporting

confidence: 85%

Effect of Signal Peptide on Stability and Folding of Escherichia coli Thioredoxin

et al. 2013

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“…[13][14][15][16][17][18] The reason for disulfide stabilization has been attributed to both entropic and enthalpic effects. One theory suggests that a decrease in entropy of the unfolded state is causing destabilisation of this state compared with the folded state.…”

Section: Introductionmentioning

confidence: 99%

“…Selecting new positions for introduction of an additional stabilizing disulfide bond is not trivial as there are strict stereochemical requirements for the relative position and orientation of the two involved Cys. 14,23 The introduction of a fourth disulfide into the relatively small insulin molecule poses a challenge to the yeast expression system. In the yeast expression system insulin is folded and the three existing disulfide bonds are formed during expression.…”

Section: Introductionmentioning

confidence: 99%

Insulin analog with additional disulfide bond has increased stability and preserved activity

et al. 2013

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Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Ca-Ca distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the Bchain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

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“…The activity of Trx was assayed by the insulin aggregation assay (Das et al 2007). Phosphatase activity of Drosophila PTPs purified by TPP of 8 M urea solubilized inclusion bodies (dark gray).…”

Section: Assay For Trxmentioning

confidence: 99%

“…In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al 1995;Dennison and Lovrien 1997;Jain et al 2004;Przybycien et al 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps. We have used TPP to refold 12 different proteins from inclusion bodies, namely, ribonuclease A (RNase A) (Crook et al 1960;Wagner et al 1983;Laity et al 1993;delCardayré et al 1995;Okorokov et al 1995), the first two domains of human CD4 (CD4D12) (Dalgleish et al 1984;Doyle and Strominger 1987;Sharma et al 2005), five insoluble constructs of the protein tyrosine phosphatases (PTPs) from Drosophila melanogaster (Madan and Gopal 2008), and five highly unstable and aggregation prone mutants of the E. coli proteins controller of cell division or death B (CcdB), maltose binding protein (MBP), and thioredoxin (Trx) Bajaj et al 2005;Das et al 2007). In each case we show that TPP-refolded protein is folded/active and that recovery yields and purification time are superior or comparable to conventional methods.…”

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Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

et al. 2008

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Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of threephase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.Keywords: three-phase partitioning; protein refolding; recombinant ribonuclease A; CcdB mutants; human CD4; inclusion bodies Supplemental material: see www.proteinscience.org Upon expression in Escherichia coli, many recombinant proteins form dense, insoluble aggregates called inclusion bodies (Taylor et al. 1986). It is therefore necessary to develop efficient and scalable procedures for solubilization and refolding of recombinant proteins from inclusion bodies (Misawa and Kumagai 1999;Middelberg 2002).Refolding is typically carried out using either simple dilution into appropriate refolding conditions, dialysis or on a column (Stempfer et al. 1996;Rogl et al. 1998). Finding appropriate refolding conditions is generally the most difficult step of the purification process. Aggregation and precipitation of protein during refolding are commonly encountered problems. Factorial and other screens for refolding have been developed (Hofmann et al. 1995;Armstrong et al. 1999;Bajaj et al. 2004) to facilitate the search of appropriate refolding conditions. In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al. 1995;Dennison and Lovrien 1997;Jain et al. 2004;Przybycien et al. 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps. We have used TPP to refold 12 different proteins from inclusion bodies, namely, ribonuclease A Reprint requests to: Munishwar N. Gupta, Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India; e-mail: munishwar48@yahoo.co.uk; fax: 91-011-26581073.Abbreviations: CcdB, controller of cell division or death B; CD4D12, first two domains of human CD4; C m , the denaturant concentration at which one half of the protein molecules are unfolded; GdmCl, guanidium chloride; IPTG, isopropyl b-D-1-thiogalactopyranoside; MBP, maltose binding protein; PTPs, protein tyrosine phosphatases; RNase A, ribonuclease A; SPR, surface plasmon resonance; TPP, three-phase partitioning; Trx, E. coli thioredoxin.Article and publication are at

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Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

Cited by 19 publications

References 72 publications

Effect of Signal Peptide on Stability and Folding of Escherichia coli Thioredoxin

Effect of Signal Peptide on Stability and Folding of Escherichia coli Thioredoxin

Insulin analog with additional disulfide bond has increased stability and preserved activity

Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

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