Small scale His-Tag purification under nature conditions

The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mariol@mail.ls.huji.ac.il Tel: 972-2-6586920

Contaminant Removal from Inclusion Bodies Before Solubilization

INTRODUCTION

The most common method of Inclusion Bodies (IB) preparation involves mechanical or chemical cell disruption followed by differential centrifugation to separate the dense IB from the lighter cell membrane components and soluble contaminants. Repeated washes with denaturants or detergents can considerable reduce contaminant levels before IB solubilization. These washing steps are important not only to facilitate protein purification, there are many evidences that contaminants present in preparation of IB can significantly reduce refolding yield (1).

Here we present a simple protocol to check the use of different denaturants or detergents (see example) to reduce contaminant levels before IB solubilization. According to the results you can choose more washes with different detergent or denaturants concentrations, before scale-up.

(1) Middelberg A., Preparative Protein Folding. TRENDS in Biotechnology 2002, 20 (10): 437-443 (pdf)

Reagents required

Urea
Triton X-100
n-Dodecyl-Beta-D-maltoside (Dodecyl maltoside)
n-Octyl-Beta-D-glucopyranoside (Octyl glucoside)
Lauroylsarcosine (Sarkosyl)
Lauryl Dimethyl Amine Oxide (LDAO)

Cell Disruption

Resuspend pellet of 50ml cell culture in 5ml lysis buffer.

Suggested Lysis buffer : 140mM NaCl; 2.7mM KCL; 10mM Na₂HPO₄; 1.8mM KH₂PO_4; pH 7.3 (PBS)
optional 0.02% NaN₃ (azide)
optional protease inhibitors

Optional additives to the lysis buffer
a) 1mM PMSF or protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma)
b) Dnase 100U/ml or 25-50ug/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of 10mMMgCl₂.
c) Lysozime 0.2mg/ml. Incubate 10min 4°C.
d) ßME, DTT or DTE up to 10mM for proteins with many cysteines.

Sonicate in ice bucket 3 x 10sec or more if the cells are not completely disrupted (Lysis is complete when the cloudy cell suspension becomes translucent. Avoid protein denaturation by frothing).

Spin 20min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet). Use pellet for next step. Keep sample of 40ul of supernatant for PAGE-SDS: soluble proteins

IB Washings

1) Triton X-100 wash: resuspend pellet in 2.5ml lysis buffer (without DNaseI and lysozime) + 1% Triton X-100 and mix gently 5min. Spin 20min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or IB proteins (pellet). Use pellet for next step. Keep sample of 40ul of supernatant for PAGE-SDS: Triton X-100 first wash
2) Repeat Triton X-100 wash (Keep sample of 40ul of supernatant for PAGE-SDS: Triton X-100 second wash)
3) Repeat wash without Triton X-100 and fractionate suspension before spin in several aliquots for further treatment with different washing conditions: resuspend pellet in 2.5ml lysis buffer (without DNaseI and lysozime) and mix gently 5min. Fractionate suspension in 10 plastic tubes, 0.25ml each fraction. Spin 20min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or IB proteins (pellet). Use pellet for next step. Keep sample of 40ul of supernatant for PAGE-SDS: Third wash
4) Wash each fraction with different conditions: resuspend pellet of each fraction with 0.25ml lysis buffer (without DNaseI and lysozime) + different denaturants or detergents and mix gently 5min. Spin 20min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or IB proteins (pellet). Use pellet of each fractionfor next step. Keep sample of 40ul of supernatant for PAGE-SDS: Fourth wash

Suggested conditions:
    a) 0M Urea d) 0.2% Sarkosyl        g) 1% LDAO
    b) 1M Urea            e) 0.5% Sarkosyl        h) 1% octyl glucoside
    c) 2M Urea            f) 1.0% Sarkosyl       i) 1% Dodecyl maltoside
    d) 4M Urea
You can use these suggested conditions or any other concentration used here; or a different mild detergent (like Tween 20, CHAPS, CHAPSO, etc.), or a strong detergent at low concentration (as SDS or CTAB) or any other denaturant at low concentration (as Guanidine HCl).

5) Repeat last wash: Fifth wash
6) Repeat wash without detergent or denaturant: Sixth wash
7) 8M urea solubilization: resuspend pellet of each fraction with 0.25ml lysis buffer (without DNaseI and lysozime) + 8M urea and mix gently 60min at 4C. Spin 20min 13000rpm 4°C. Separate soluble proteins (solubilized IB proteins) from pellet (unsolubilized IB). Keep sample of 40ul of supernatant for PAGE-SDS: 8M urea resuspended IB, keep sample of 40ul of resuspended pellet for PAGE-SDS: 8M urea insoluble proteins.

Analysis of results

Run PAGE-SDS of all the washings + 8M urea of soluble or insoluble proteins. Compare purity and yield of the different washing conditions, and scale-up the best option. See example

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Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem
mariol@mail.ls.huji.ac.il Tel: 972-2-6586920