GFP

GFP kdorfman Fri, 11/11/2011 - 14:25

GFP purification

GFP purification kdorfman Fri, 11/11/2011 - 14:41

sfGFP purification Reagents:

Purification Procedure

Small-Scale Culture for Screening

  1. Streak BL21 (DE3) containing pET28/sfGFP on plates and grow up overnight at 30 C.
  2. Take single colonies and inoculate 4 test tubes containing 2 mL of LB/KAN. Keep 3 more tubes just LB/KAN as blanks for OD measurements and controls.
  3. Shake at 200 rpm and 37C to mid-log growth phase, indicated by the OD600 nm of 0.6 to 0.7. (Referenced against LB/KAN). This takes approximately 3 hours.

Small-Scale Induction

  1. Split 2 mL cultures into two 1-mL cultures and induce one of the cultures with 1 L of 1 M IPTG. Grow the other 1-mL cultures un-induced for comparison.
  2. After IPTG has been added, incubate for 1 h. Small-Scale Screening
  3. Take 500 uL of the induced and un-induced cultures. Centrifuge the cells in 1.5 mL microcentrifuge tubes for 15 min at 13000 rpm (maximum).
  4. Discard the supernatant and suspend pellets in 20 uL of de-ionized water and 20 uL of gel loading buffer for SDS-PAGE.
  5. Boil for 5 min. and check for induction using SDS-PAGE (12.5% gel) in a side-by-side comparison of induced and un-induced cultures to ensure that sfGFP is produced.

Large Scale Cultures and Purification

Day 1

In the latter part of the day set up a 50 mL overnight culture of GFP in BL21(DE3) to obtain a dense culture

  • Add 50 uL of 1000x KAN to 50 mL LB
  • Inoculate culture medium with GFP BL21(DE3) cultures that produced GFP the best (inoculate with un-induced matched sample)
  • Shake at ~200-275 rpm and 30C

Day 2

  1. Inoculate 0.5 or 1 L LB cultures containing 50 ug/mL LB/KAN with 5 to 15 mL of the overnight culture and grow at 30C with shaking at 200 rpm until the OD600 equals 0.6-0.7. Add IPTG to a final concentration of 1 mM and continue the growth for 3 hrs.
  2. Harvest the cells by centrifugation at 3,000 rpm for 30 min. and discard supernatant.
  3. Resuspend the pellet in Buffer A. The final volume of Buffer A should be 25 mL for a cell paste obtained from a 1 L culture.
  4. Freeze the sample and store overnight at -80 C

Day 3

  1. On the following day freeze/thaw the cells 3 times to break up the cell matrix.
  2. Add lysozyme (10 mg/50 mL) and shake gently for 20-30 min at 25C.
  3. Sonicate the cell slurry at 35% power and 35% duty cycle, on ice with occasional stirring, until the solution turns viscous and then returns to a less viscous consistency (about 15 minutes).
  4. Centrifuge the sample at 10,000 rpm at 4C for 1 h.
  5. Collect the supernatant and centrifuge it at 40,000 rpm at 4C for 1 h in Ti 70 rotor. Save the supernatant for affinity column purification.

Preparation of the Ni-NTA column

Washing steps of column before sample run

  • 15 mL 20% of ethanol.
  • 15 mL distilled water
  • 15 mL freshly prepared 0.1 M NiSO4
  • 15 mL of distilled water
  • 15 mL of Buffer A
    • Load the column with sample and adjust the rate at 30/25 mL sample using peristaltic pump or 1 mL/min in HPLC.
    • Wash the column using 3 bed volumes of Buffer B or until the UV280 signal returns to baseline.
    • Elute histidine tagged protein with Buffer C.
    • After using the column, wash it with 5 to 10 column volumes of 0.05 M EDTA, distilled water and 20 %ethanol respectively (Column regeneration).

Protein Dialysis

  • Transfer the sample eluted from the Ni-NTA column using Buffer C to dialysis tubing with a 10,000 MWCO.
  • Dialyze the sample using about 800 mL of Buffer A 3 times. The first round of dialysis is carried out overnight; the 2nd and 3rd rounds are carried out for 2-3 h periods.
  • Assess purity of sample using SDS–PAGE (12.5% acrylamide gel).
  • Determine concentration by absorbance at 485 nm
    Extinction coefficient = 8.33 x 10^4 M-1cm-1 (ref. 1).
  • Flash Freeze in LN2 and store @ -80C.

Plasmid sequence

Plasmid sequence kdorfman Fri, 11/11/2011 - 15:09

pET 28/sfGFP

The sfGFP expression plasmid is based on the pET28 expression vector with Kanamycin resistance. The host strain is BL21(DE3).

Pe´delacq, Cabantous, Tran, Terwilliger and Waldo (2006) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotech. 24, 79-88.

https://www.ncbi.nlm.nih.gov/pubmed/16369541

pET 28/sfGFP sequence (5975 bp)

The sfGFP coding region is bold (741 bp).

TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACC GCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCT TTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCT CGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA ACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAAT GTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCT TAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTT GAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTA TCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTA TCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCAT TCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAA TGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATA CCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATG CTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTG TCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATT TAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATG TAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGAC CCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAAC TGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTA TGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCT CTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATT CTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATT TCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACT CCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAG GTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGAT TCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTC TGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATT TCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAA CATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAA TCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCC TGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGG AAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCT CGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAG GAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTG GTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGA TCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTAC GAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAG CTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGG CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGG CAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCC AGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGT ATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGC CATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGA AAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATT TATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTG GTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTG ATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGG CATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGC CGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCG CGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCA GCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGC TTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAA GAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCT CTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCT CTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGC AAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACG CCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGG CGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTC GATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAA TTTTGTTTAACTTTAAGAAGGAGATATACATATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCA ATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGaGGAGAGGGTGAAGGTGATG CTACAaACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACT TGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCAcATGAAACGGCATGACTTT TTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAcCTACA AGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTT TAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTtTAACTCACACAATGTATACATCACG GCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACgTTGAAGATGGtTCCGTTC AACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTA CCTGTCGACACAATCTGtCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTT GTAACTGCTGCTGGGATTACACATGGCATGGatGAGCTCTACAAAGGATCCCATCACCATCACCATCACT AACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGC TGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG CTGAAAGGAGGAACTATATCCGGAT

BL21(DE3)

BL21(DE3) kdorfman Tue, 10/23/2018 - 15:38

Info on growth of BL21(DE3):

https://link.springer.com/article/10.1007%2Fs00253-006-0554-8#Sec2

Table 1 The maximum optical density and doubling time (DT) of E. coli BL21 (DE3) aerobic batch cultures in different minimal media supplemented with 0.4% glycerol

Medium DT (min) Max OD600nm
M9 102±9 1.2±0.19
N−C− 78±6 1.6±0.14
LR 66±3 3.1±0.14
M9 + Fe2+ 71±1 2.9±0.13
M9 + PO4 3− 71±2 2.2±0.08

N−C− minimal medium contained 10 mM of NH4Cl.

FeSO4·7H2O (0.01 mM) and KH2PO4 (107 mM) were added to M9 minimal medium to supplement it with iron and additional phosphate, respectively.

LeMaster and Richards minimal medium (LR medium) consisted of a mixture of buffer salts (KH2PO4 24.0 g l-1 and NaOH 1.0 g l-1), and mineral salts [(NH4)2SO4 1.675 g l-1, MgSO4·H2O 0.3 g l1, FeSO4·7H2O 3.0 mg l-1, concentrated H2SO4 10 μl l-1], prepared as 10× and 100× concentrates, respectively, and mixed as buffer salts–mineral salts–water in the ratio 10:1:89 (LeMaster and Richards 1982; Paliy et al. 2003).

Values are shown as arithmetic mean±standard error of the mean (n=3).

Only two replicates were used for N-C- and LR

Protein sequence

Protein sequence kdorfman Fri, 11/11/2011 - 15:10

sfGFP protein sequence (241 amino acids), MW 27748 g/mol

MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSHHHHHHStop

The sequence was translated using the EXPASY proteomic server (http://au.expasy.org/tools/dna.html).