In vitro folding of inclusion body proteins

Rudolph, Rainer; Lilie, Hauke

doi:10.1096/fasebj.10.1.8566547

Cited by 619 publications

(432 citation statements)

References 58 publications

Supporting

Mentioning

422

Contrasting

Unclassified

Order By: Relevance

“…As shown for other receptors, e.g. the N-terminal domain of the ␣7 AChR (13), proteins from inclusion bodies may properly refold, but the refolding procedure does not necessarily result in a high percentage of correctly folded protein (35). While we made considerable efforts to avoid inclusion body formation by testing different E. coli strains and varying the expression conditions, we never obtained sufficient amounts of the target protein in its soluble form (not shown).…”

Section: Resultsmentioning

confidence: 85%

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

Breitinger

Bauer

Fahmy

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding ␣-and gephyrin anchoring ␤-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (␣1-(1-219)) and the large intracellular TM3-4 loop (␣1-(309 -392)) of the human GlyR ␣1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested ϳ25% ␣-helix and ϳ48% ␤-sheet for the extracellular domain, while no ␣-helices were detectable for the TM3-4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of ϳ5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of ϳ49 kDa indicated oligomerization in a defined native structure. As shown by [ 3 H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.The inhibitory glycine receptor (GlyR) 1 is a neurotransmitter-gated ion channel, mediating rapid synaptic transmission in the central nervous system (1-3). The GlyR is a member of the ligand-gated ion channel superfamily, which also includes the nicotinic acetylcholine receptor (nAChR), serotonin 5-HT 3 receptor, as well as GABA A/C receptors. Members of this protein family share a quaternary structure of five subunits surrounding a central ion-conducting pore (4 -6). The GlyR isoform prevalent in adult mammalian central nervous system is thought to comprise three ␣1-subunits and two structural ␤-subunits, which are thought to contribute to synaptic anchoring of receptor complexes. Generally, GlyR ␣-subunits possess the ability to form functional homomeric receptor channels. The subunit topology of the nAChR superfamily, as deduced from hydropathy analysis, consists of a large N-terminal extracellular domain, followed by four hydrophobic stretches of sufficient length to span the plasma membrane, and the extracellular C terminus (7). Of all transmembrane regions, TM2 forms the inner lining of the central ion channel. A large cytosolic loop, flanked by TM3 and TM4, is thought to mediate intracellular interactions.

show abstract

Section: Resultsmentioning

confidence: 85%

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

Breitinger

Bauer

Fahmy

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Because aggregation is typically at least second order in protein concentration (16,17), to prevent reaggregation, the protein typically must be diluted to between 10 and 100 g/ml before the chaotrope is removed. Unfortunately, achieving acceptable refolding yields, if possible, usually requires screening of a large number of refolding conditions (18,19). Frequently, after extensive research, refolding yields remain too low to justify commercial product recovery with chemical refolding methods.…”

mentioning

confidence: 99%

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

John¹,

Carpenter²,

Balny³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Two different types of insoluble, non-native aggregates of recombinant human growth hormone were formed by agitation in buffer or buffer containing 0.75 M guanidine HCl (GdnHCl) and characterized by infrared and second derivative UV spectroscopies. The degree of secondary structural perturbation was greater in the aggregates formed in 0.75 M GdnHCl. Both aggregate types were dissolved and refolded using high hydrostatic pressures in combination with either elevated temperature or non-denaturing levels of guanidine HCl or urea. The effects of a range of temperature, pressure, and chaotrope concentrations were tested and led to optimal conditions that approached 100% yield of native protein. The aggregates formed in 0.75 M GdnHCl required higher concentrations of urea or GdnHCl, or higher temperature or pressure for a yield equivalent to that for aggregates formed in buffer alone. Investigation of the effects of pressure, temperature, and chaotrope on unfolding of rhGH documented that under conditions used for optimal high pressure disaggregation and refolding, the native state is greatly favored thermodynamically (e.g. 25 kJ/mol at 2000 bar and 0.75 M GdnHCl). Dissolution of aggregates under pressure is a kinetically limited process. Comparison of refolding yields in GdnHCl and urea solutions suggest that pressure effects on electrostatic interactions do not dominate pressure effects on disaggregation. We suggest that non-native hydrogen bonds between protein molecules within aggregates of recombinant human growth hormone are responsible for the rate-limiting kinetic barrier in pressure-induced disaggregation.

show abstract

“…The use of Tris buffer at a concentration of at least 0.4 M was found to be necessary for obtaining the refolded PBP1 derivative. This has been reported for several test proteins expressed in E. coli [23]. The effect of -arginine to increase yields of in itro folding of proteins is also well documented, although not clearly understood.…”

Section: Solubilization Of [∆M1-r314]pbp1mentioning

confidence: 80%

Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

Mahapatra

Bhakta

Ahamed

et al. 2000

Biochemical Journal

View full text Add to dashboard Cite

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627–4630]. PBP1-Xaa–β-lactamase fusions generated periplasmic β-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [∆M1–R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [∆M1–R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.

show abstract

In vitro folding of inclusion body proteins

Cited by 619 publications

References 58 publications

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

Contact Info

Product

Resources

About