Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:4081713 |
SGC Construct ID: AASDHPPTA-c003 |
GenBank GI number: gi|20357568 |
Vector: pCOEX-1 |
Tags and additions (His 6 + TEV cleavage site):
mgsshhhhhhhssgrenlyfqgh |
Host : E.coli BL21 DE3-R3 |
Construct coding sequence:
mgsshhhhhhhssgrenlyfqghMEGVRW
AFSCGTWLPSRAEWLLAVRSIQPEEKERI
GQFVFARDAKAAMAGRLMIRKLVAEKLNI
PWNHIRLQRTAKGKPVLAKDSSNPYPNFN
FNISHQGDYAVLAAEPELQVGIDIMKTSF
PGRGSIPEFFHIMKRKFTNKEWETIRSFK
DEWTQLDMFYRNWALKESFIKAIGVGLGF
ELQRLEFDLSPLNLDIGQVYKETRLFLDG
EEEKEWAFEESKIDEHHFVAVALRKPDGS
RHQDVPSQDDSKPTQRQFTILNFNDLMSS
AVPMTPEDPSFWDCFCFTEEIPIRNGTKS |
Growth medium, induction protocol: Medium: TB + 34 µg/ml Chloramp. Cells were grown in 1 liter TB in 2.5-L baffled flasks, which was inoculated with 10 ml overnight culture. The culture was grown at 37°C to OD=2.3, and transfered to 25°C. 1 mM IPTG was then added, and incubation continued for 4 hours. The cells were then collected by centrifugation and frozen at -80°C. |
Extraction buffer, extraction method: Lysis buffer: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, Complete® protease inhibitors (1 tablet/50 ml). |
Frozen cell pellets were thawed on ice over night and resuspended in a total volume of 40 ml lysis buffer, the cells were disrupted by high pressure (20 psi) followed by sonication. |
Nucleic acids and cell debris were removed by adding 0.15% PEI from a 5% (w/v) stock, stirring for 15 minutes, then centrifugation for 20 minutes at 40,000xg. The supernatant was then further clarified by filtration (0.45 µm). |
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham) |
Buffers: Lysis buffer: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP. Wash buffer: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 50 mM imidazole, 0.5 mM TCEP. Elution buffer: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 250 mM imidazole, 0.5 mM TCEP. |
Procedure: The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of Lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected. |
Column 2 : Gel filtration, Hiload 16/60 Superdex 200 prep grade 120 ml (GE Healthcare) |
Buffers : GF buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP. |
Procedure: The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions. |
Concentration : In vivaspin 6 ml 10 K. Concentration determined from the absorbance at 280 nm using the extinction coefficient and the nanodrop method. |
Concentration : 19.6 mg/ml |
Enzymatic treatment : no cleavage |
Mass spectrometry: observed mass corresponds to predicted protein sequence |
Target ID: ACP domain of FASNA |
Entry clone source: An ACP fragment was amplified from a human liver cDNA libaray and cloned into a bacterial expression vector. Site directed mutagenesis of the phosphopantetheinyl acceptor site (Ser39) was exchanged to Ala using PCR . |
Vector: pQE 80 L (Qiagen). |
Sequence and Tags/ additions:
(His-tag and TEV protease site in lowercase)
mrgshhhhhhgsdydipttenlyfqgtRD
RDSQRDLVEAVAHILGIRDLAAVNLDSSL
ADLGLDALMSVEVRQTLERELNLVLSVRE
VRQLTLRKLQELSSKADEASELACPTPKE |
Host : E. coli BL21-codon plus. |
Growth medium, induction protocol: Cells were grown at 37°C in LB medium +100µg/ml carbenicillin to A 600 of 0.5-0.7 and induced with 1 mM IPTG for 3 h. |
Extraction buffer, extraction method: Cells were suspended in 0.25 M potassium phosphate buffer, pH 7, containing 10% glycerol 5 mM mercaptoethanol and protease inhibitors (leupeptin 5µg/ml, trans-epoxysuccinyl LGB 10µM, pepstatin 1µg/ml, and antitrypsin 5µg/ml final concentration), and lysed by four disruption cycles in a microfluidizer. The lysate was centrifuged at 50,000 g for 1 h at 4 °C, the supernatant was collected and filtered (0.4µ). |
Column 1 : Ni-NTA affinity |
Procedure: The soluble cell extract was loaded onto a NiNTA column at 0.5 ml/min and the column eluted successively with 10 vol buffer A ( 0.1 M potassium phosphate buffer, pH 7/2 mM mercaptoethanol, 10% glycerol) and 10 vol buffer B (Buffer A containing 125 mM imidazole. fractions were analyzed by SDS - PAGE and those containing ACP were pooled, DTT was added to 2 mM, final concentration and the sample was dialyzed overnight against 50 mM Tris-HCl pH 7.5/10% glycerol/1 mM DTT. |
Column 2 : Resource-Q anion exchange |
Procedure: The dialysate was filtered (0.4 µ) and applied at 2-ml per min to a column (5 ml) of Resource-Q equilibrated with 50 mM Tris-HCl pH 7.5/10% glycerol/1 mM DTT and eluted with a gradient containing 0-0.25 M NaCl. The major A280-absorbing zone was collected and a portion was subjected to cleavage with TEV protease. |
Concentration : 10 mg/ml |
Enzymatic treatment : ACP was treated with AcTEV protease (Invitrogen), 10 U/mg ACP for 24 h at 20°C followed by 40 h at 4°C, then reapplied to a Ni-NTA affinity column. This time the ACP , lacking the His-tag, eluted in the wash buffer while the AcTEV protease and the cleaved N-terminal peptide remained bound to the column. The sample was then dialyzed against 50 mM Tris pH 7.4/0.3M NaCl/1 mM DTT/10% glycerol and concentrated using a Vivaspin device (5k Da cutoff). |
Mass spec characterization : Both the purified TEV-cleaved and not-cleaved ACPs were homogeneous, as evaluated by SDS - PAGE and Coomassie staining. The molecular masses of the TEV-cleaved and not-cleaved ACPs, determined by MALDI-MS, were 10,053.4 and 13,053.3 Da, respectively, close to the expected values. Protein concentration was estimated using the BioRad BCA method with bovine serum albumin as standard. |
