Refolding Record:
| Protein | |
|---|---|
| Protein Name | Trypsin Inhibitor (TI) |
| Abbreviated Name | TI |
| SCOP Family | Proteinase/alpha-amylase inhibitors |
| Structure Notes | |
| Organism | Corn |
| UniProt Accession | P01088 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 133 |
| Molecular Weight | 14188.3 |
| Pi | 7.62 |
| Molecular Weight | 14188.3 |
| Disulphides | 5 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMSAGTSCVPGWAIPHNPLPSCRWYVTSRTCGIG
PRLPWPELKRRCCRELADIPAYCRCTALSILMDGAIPPGPDAQLEGRLEDLPGCPREVQR
GFAATLVTEAECNLATIS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chen ZY, Brown RL, Lax AR, Cleveland TE, Russin JS. (1998) Applied and Environmental Microbiology, 65, 1320-1324 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 6 h |
| Expression Vector | pET-28b |
| Expression Protocol | Overexpression of the TI gene in E. coli and purification technique. The complete coding region of mature corn 14-kDa TI cDNA (GenBank accession no. X54064) (19) was amplified from plasmid pT7-7 with Taq polymerase by using the primer pair 2041 (5′ GAGCTCTTACTTGGAGGGCATCGTTCCGC) and 2164 (5′ CATATGAGCGCCGGGACCTCCTGC) with mismatches (underlined) to introduce an NdeI (5′ end) or SacI (3′ end) restriction site. After cloning into TA cloning vector pCRII (Invitrogen, Carlsbad, Calif.) and complete sequence analysis, the amplified 0.4-kb PCR product was subcloned into the unique NdeI and SacI sites of an E. coli overexpression vector, pET-28b (Novagen, Madison, Wis.). Positive clones were identified by using PCR according to the manufacturer’s instructions. The correct in-frame fusion of the construct was verified by DNA sequencing of positive transformants before it was transformed into an E. coli BL21 (DE3) expression host. TI expression was induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM as previously described (5). The overexpressed TI was predicted to be 16.5 kDa, containing a vector His tag and a thrombin cleavage site at the N terminus (MGSSHHHHHHSSGLVPRGSHM) followed by the complete mature TI (127 amino acid residues) (19).E. coli cells overexpressing TI were harvested from a 500-ml culture after 6 h of induction, washed twice with 50 mM Tris-HCl (pH 8.0), and then resuspended in 10 ml of the same buffer. The cells were ultrasonically disrupted on ice with pulses delivered intermittently for 6 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Tris-Hcl 50 mM, pH 8.0 |
| Solubilization Buffer | 50 ml of 50 mM Tris-HCl (pH 8.0) containing 6 M urea |
| Refolding Buffer | 50 ml of 50 mM Tris-HCl (pH 8.0) containing 6 M urea and 140 mM β-mercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 140 mM |
| Refolding Protocol | Inclusion bodies were recovered by centrifugation (18,000 × g, 20 min) and washed twice with the same buffer. The supernatant was saved as the water-soluble fraction. Inclusion bodies were then resuspended in 100 ml of 50 mM Tris-HCl (pH 8.0) containing 6 M urea, and urea-soluble proteins were separated from the urea-insoluble fraction by centrifugation (18,000 × g, 20 min) and saved as the urea-soluble fraction. This step was repeated three times. The resulting urea-insoluble fraction was dissolved in 50 ml of 50 mM Tris-HCl (pH 8.0) containing 6 M urea and 140 mM β-mercaptoethanol. After insoluble cell debris was removed by centrifugation, proteins in the supernatant (β-mercaptoethanol-soluble fraction) were subjected to refolding with cystamine as described previously by Kohno et al. (10). (The protein was further diluted in this solution to a final |
| Refolding Assay | enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | cystamine |
| Additives Concentration | 250 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |