Large-scale purification of recombinant proteins



Optimizing expression conditions

Before starting the large-scale purification of a protein from 1-5 l of culture, the conditions for the expression have to be optimised. The expression levels of the recombinant proteins can vary over several orders of magnitude, depending on the following parameters.
  • Temperature: The soluability of the protein may be improved by culturing at low temperature, usually 18C. Expression is induced then at higher density (OD=0.8) and expression period is longer, often overnight.
  • Concentration of IPTG: Although the expression does not depended linearly on the IPTG concentration, at 0.1 mM instead of the usual 1 mM expression is slowed down. This may result in a higher soluability of the expressed protein.
  • E. coli strain. Expression levels may differ like black and white in two strains. However it is impossible to predict, which one is better. Use strains supplemented with plasmids carrying genes for rare tRNA, if these codons are present in your construct.

Expression

  • transform BL21DE+rosetta(chlorampenicolR) with the plasmid plate on a selective plate and grow in a 100 ml culture. Do not store the transformed cells for longer than one week. BL21 E. coli are genetically pretty instable.
  • inocculate 1000 ml of prewarmed LB+Amp with the overnight culture to OD=0.1, grow until OD=0,5 at 37°C
  • take a 1 ml sample, spin for 1 min at 13 k, dissolve the pellet in Lämmli (add 50 ul, if OD=0.5)
  • add IPTG to 0,1 - 1 mM IPTG
  • shake at 18°C overnight or 1-4 h at 37°C
  • measure OD600, take a 1 ml sample (total extract)
  • add protease inhibitors: 1 mM PMSF, 250 uM ortho Phenanthrolin, 1 mM Benzamidine
  • collect cells (4-5k, 10 min)
  • thoroughly suspend the cells in Lysis buffer. Use 1 ml for 100 OD, e. g. 10 ml for 1000ml culture of OD=1. For very dense lysates freeze and thaw the pellet and add DNase and lysozyme
  • lyse cells with the microfluidizer in Ficner's lab or sonicate
  • spin 20 min, 12 k (pellet is relatively large), transfer supernatant with a pipette to a new vial, take a sample of the pellet (insoluable fraction)
  • spin again, 20 min, 12k, or spin in the ultracentrifuge, transfer supernatant (must be clear) to an new vial

preparation of the column

  • Ni-HiTrap:(PDF-manual) wash column with 50 mM EDTA (10 volumes) to strip of the Ni2+
  • wash  with 10 volumes of water
  • wash with 10 volumes of 100 mM NiSO4 to load the  column, wash with water, and wash with lysis buffer
  • make sure before applying the sample that all tubing contains lysis buffer!
  • GSH-HiTrap(PDF-manual) equilibrate the column with lysis buffer (10 volumes)

    • column chromatography

  • apply the clear (!) supernatant to the column (0,5-1 ml/min)
  • collect flow through, collect a sample (unbound)
  • wash column with washing buffer (> 10 volumes), until the A280 does not change anymore For large preparations you may use a second -more stringent- washing buffer.
  • elute with elution buffer, collect fractions of 0,5 column volumes
  • drop 1 µl of each fraction on a small stripe of nitrocellulose, fix with destain solution, stain with Amidoblack for 5 min, wash with tap water to destain
  • pool fractions containing protein
  • dialyse or perform buffer exchange with desalting columns (e. g. NAP-10 or G25
  • determine protein concentration by Bradford assay
  • add glycerol to 50% or sucrose to 200 mM
  • snap freeze aliquots in liquid nitrogen and store at -70°C

elution with a gradient

  • load lysate on the column
  • wash with lysis buffer until the OD does not change anymore (>20 vol)
  • establish a gradient with A:  lysis buffer and B: 1 M imidazol (pH 8), elute over 20 volumes

SDS-PAGE:

total extract:  suspend cells from 1 ml culture in 50 µl for OD600=1, load 5 µl



buffer:

Type Ni2+ column GSH column
Lysis 20 mM Na-phosphate, pH 8,
500 mM NaCl
20 mM imidazol
 50 mM Tris/HCl pH8
100 mM NaCl
1 mM DTT
or PBS
Wash 20 mM Na-phosphate pH 8
500 mM NaCl
40 mM imidazol
 50 mM Tris/HCl pH8
500 mM NaCl
1 mM DTT
Elution 20 mM Na-phosphate, pH 8
500 mM NaCl
250 mM imidazol
 50 mM Tris/HCl pH 8
50 mM NaCl
10 mM Gluthathion


GSH elution buffer
prepare aliquots of 5 ml 100 mM Gluthathion and freeze at -20°C. Only before use dilute these aliquots into 50 ml elution buffer. Check pH! Do not store the 1x elution buffer.

stripping buffer
50 mM EDTA, pH 8


loading buffer
100 mM NiSO4

IPTG
1 M IPTG in water

PMSF
1 M PMSF in 2-propanol,
store at -20¡C

ortho-Phenanthrolin
100 mM ortho-Anthrolin in 2-propanol,
store at -20¡C

Benzamidin-HCl
use about 0.15 g/l (1 mM) final concentration
store powder at 4¡C