Refolding Record:
| Protein | |
|---|---|
| Protein Name | Apolipoprotein(a) |
| Abbreviated Name | Lp(a) |
| SCOP Family | Kringle Modules |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P08519 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Kringle 4 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 115 |
| Molecular Weight | 12507.7 |
| Pi | 5.47 |
| Molecular Weight | 12507.7 |
| Disulphides | 3 |
| Full Sequence |
APTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kang KY, Kim SG, Kim WK, You HK, Kim YJ, Lee JH, Jung KH, Kim CW. (2006) Protein Expression and Purification, 45, 216-25 |
| Project Aim | Purification & characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 30.0 |
| Expression Time | overnight |
| Expression Vector | pET11a |
| Expression Protocol | Expression and fermentation The LK68 was cloned into the pET11a plasmid vector (Novagen, Darmstadt, Germany), under the control of the T7 promoter, and then introduced to E. coli BL21 (DE3) [16]. The transformed E. coli was then cultured in a 500 ml flask containing 200 ml LB media (Bacto tryptone (Difco, USA), 10 g/L; yeast extract (Difco, USA), 20 g/L; and NaCl, 5 g/L) in an incubator with agitation at 30 °C overnight, and then transferred to a 5-L fermentor (BiofloIII, New Brunswick Sci. Co., USA) containing 2 L of starting media (glycerol 20 g/L; other media components), for the large-scale culture. In order to foster the growth and expression of LK68, lactose feed medium mixed with glycerol (glycerol, 500 g/L; lactose, 10 g/L; and other media components) was continuously supplied as a dual source of carbon and inducer in a dissolved oxygen (DO)-stat manner, which turns on the feed pump when the DO level increased above 50% of saturation and turns it off when the DO level decreased below 50% [18]. The quantity of LK68 in the fermentation broth was assessed by running the total fermentation broth (20 μl) on Tris–glycine, 4–20%, SDS–PAGE, and comparing the LK68 band in the fermentation broth with the band formed by the standard LK68, which was finally purified and quantified via the Bradford method, using BSA as a standard, after the gel had been stained with Coomassie’s brilliant blue R250. In order to quantify the LK68 on the gel, we scanned the gel with ScanJet (Hewlett–Packard, USA) and the scanned images were analyzed with an analysis program (Biomed Instruments, Zeineh Programs, Universal Software, USA). |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | distilled water |
| Solubilization Buffer | 8 M urea, 50 mM Tris, 50 mM sodium chrolide, 10 mM EDTA, and 100 mM 2-mercaptoethanol, pH 8.8 |
| Refolding Buffer | 2.5 M urea, 20 mM sodium phosphate, 50 mM sodium chloride, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione, at a pH of 8.8 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.8 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/0.2 mM |
| Refolding Protocol | About 100 g of inclusion bodies was dissolved in 2 L of urea solution (8 M urea, 50 mM Tris, 50 mM sodium chrolide, 10 mM EDTA, and 100 mM 2-mercaptoethanol, pH 8.8) with agitation overnight at 4 °C. The LK68 in the dissolved inclusion bodies was refolded via two-step dilutions. In the first step, the LK68 in the urea solution was diluted 10-fold with 2.5 M urea, 20 mM sodium phosphate, 50 mM sodium chloride, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione, at a pH of 8.8, then stirred overnight at 4 °C. In the second step, the LK68 refolded during the first step was diluted twofold with 20 mM sodium phosphate and 50 mM sodium chloride, at a pH of 8.8, and then stirred overnight at 4 °C for further refolding. The LK68 refolded by two dilution steps was then acidified with hydrochloric acid to a pH of 4.0, and filtered through Sartopure PP2 (Sartorius, Goettingen, Gremany) filters, with 5 μm pores, for clarification prior to column chromatography. |
| Refolding Assay | HPLC,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 30% |
| Purity | n/a |
| Notes | n/a |