Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin

Tikhonov, Roman V.; Pechenov, Sergey E; Belacheu, Irina A.; Yakimov, S. A.; Klyushnichenko, Vadim; Tunes, Heloisa; Thiemann, J. E.; Vilela, Luciano; Wulfson, A. N.

doi:10.1016/s1046-5928(02)00531-4

Cited by 14 publications

(9 citation statements)

References 23 publications

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“…This hydrophobic character must have contributed to protein stability. Tikhonov et al constructed four fusion protein-S-sulfonates linked by arginine and reported that the type and the size of the leader peptide does not have an essential influence on the folding efficiency (Tikhonov et al, 2002). In our study, we found that the type and the size of leader peptide change the hydrophobicity and pI of the protein and that this change affects the pH precipitation profile of folded proinsulin.…”

Section: Folding Of the Fusion Proteinssupporting

confidence: 46%

“…2). This means that the sensitivity against redox reagent was not only affected by the hydrophobicity of protein, but also by the protein concentration of refolding and the location of (His) 10 in leader peptide (Tikhonov et al, 2002). If the purity of T10R is similar or higher than H27R, the sensitivity of T10R against redox agent might be higher than that of the H27R.…”

Section: Folding Of the Fusion Proteinsmentioning

confidence: 99%

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Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Min

Son

Kim

et al. 2011

Journal of Biotechnology

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Section: Folding Of the Fusion Proteinssupporting

confidence: 46%

Section: Folding Of the Fusion Proteinsmentioning

confidence: 99%

Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Min

Son

Kim

et al. 2011

Journal of Biotechnology

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“…The recombinant IGF1s were generally produced as inactive and intracellular inclusion bodies in E. coli, but few successes have been announced concerning the soluble and active expression of IGF1 improved by the fusion of affinity tags [9,10]. A commercially available analog of IGF1 (Long-R3-IGF1) is sold by GroPep Pty Ltd (Adelaide, Australia) as a supplement for mammalian cell culture and is also known to be accumulated as inclusion bodies when expressed in recombinant E. coli [11].…”

Section: Introductionmentioning

confidence: 99%

Effects of l-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

Choi

Park

Lee

et al. 2011

Bioprocess Biosyst Eng

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Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGF1). Fed-batch fermentation of E. coli TG1/pAPT-K6Ub-IGF1 resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM L-arginine, and 4 °C was chosen to optimize the refolding of K6Ub-IGF1, and 240 mg/L of denatured K6Ub-IGF1 was refolded with a 32% yield. The positive effect of L-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGF1 from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGF1, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that L-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.

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“…Therefore, refolding attempts can be undertaken directly after solubilization of the inclusion bodies. Some reports, however, claim higher refolding yields when the solubilized inclusion body proteins are purified prior to the refolding attempt [36,39,42,43]. Additional purification has been recommended when the protein of interest represents less than 2–5% of the total cell protein [26] or less than 2/3 of the total inclusion body protein [42].…”

Section: Introductionmentioning

confidence: 99%

“…These problems can be overcome by modifying the reduced thiol groups in the unfolded protein, either by S-sulfonation [39,128,135,136] or by transforming the free cysteines into mixed disulfides with the oxidized form of a thiol reagent ( e.g . glutathione) [128,137].…”

Section: Introductionmentioning

confidence: 99%

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2004

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Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix-or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low-and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during in vivo folding such as processes based on natural and artificial chaperones and propeptide-assisted protein refolding are presented. Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures.

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Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin

Cited by 14 publications

References 23 publications

Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Effects of l-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

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