8. It is possible to exchange the detergent to Lauryl Maltose
Neopentyl Glycol (LMNG, Anatrace) at this step. To do this,
replace DDM in the Ni-NTA Wash buffer with 0.1% w/v
LMNG and in all other buffers with 0.03% w/v LMNG. In
the final size exclusion chromatography step, the concentration
of LMNG in the S6 buffer can be further lowered to 0.002%
w/v LMNG. The critical micelle concentration of LMNG is
0.001% w/v in water.
9. The molecular weight of holo-translocon is 240 kDa and the
extinction coefficient is ~500,000 M
À1 cm
À1 at 280 nm.
10. Equilibrate the desalting column during the Ni-NTA wash
steps.
11. The maximum pressure of the desalting column is 0.3 MPa.
12. Very important: Quickly proceed from step 14 of Subheading
3.4 to the desalting step to avoid aggregation of the holotranslocon sample in the Ni-NTA elution buffer due to high
imidazole concentration.
13. Superose 6 maximum pressure is 1.5 MPa; recommended flow
rate is 0.4 mL/min.
Acknowledgments
This work is supported through a BBSRC Responsive Mode Grant
(BB/P000940/1) and a Wellcome Trust Investigator Grant
(210701/Z/18/Z). We would like to thank Kyle Powers, Flint
Stevenson-Jones, and Josh Bufton for critically reading the
manuscript.
References
1. Babu M, Vlasblom J, Pu S et al (2012) Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae. Nature
489:585–589
2. Hattab G, Warschawski DE, Moncoq K et al
(2015) Escherichia coli as host for membrane
protein structure determination: a global analysis. Sci Rep 5:12097
3. Miroux B, Walker JE (1996) Over-production
of proteins in Escherichia coli: mutant hosts that
allow synthesis of some membrane proteins
and globular proteins at high levels. J Mol
Biol 260:289–298
4. Wagner S, Klepsch MM, Schlegel S et al (2008)
Tuning Escherichia coli for membrane protein
overexpression. Proc Natl Acad Sci U S A
105:14371–14376
5. Studier FW (2005) Protein production by
auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
6. Zorman S, Botte M, Jiang Q et al (2015)
Advances and challenges of membrane-protein
complex production. Curr Opin Struct Biol
32:123–130
7. Li GW, Burkhardt D, Gross C et al (2014)
Quantifying absolute protein synthesis rates
reveals principles underlying allocation of cellular resources. Cell 157:624–635
8. Smolke CD, Keasling JD (2002) Effect of gene
location, mRNA secondary structures, and
RNase sites on expression of two genes in an
engineered operon. Biotechnol Bioeng
80:762–776
Production of Multi-subunit Membrane Protein Complexes
15
Neopentyl Glycol (LMNG, Anatrace) at this step. To do this,
replace DDM in the Ni-NTA Wash buffer with 0.1% w/v
LMNG and in all other buffers with 0.03% w/v LMNG. In
the final size exclusion chromatography step, the concentration
of LMNG in the S6 buffer can be further lowered to 0.002%
w/v LMNG. The critical micelle concentration of LMNG is
0.001% w/v in water.
9. The molecular weight of holo-translocon is 240 kDa and the
extinction coefficient is ~500,000 M
À1 cm
À1 at 280 nm.
10. Equilibrate the desalting column during the Ni-NTA wash
steps.
11. The maximum pressure of the desalting column is 0.3 MPa.
12. Very important: Quickly proceed from step 14 of Subheading
3.4 to the desalting step to avoid aggregation of the holotranslocon sample in the Ni-NTA elution buffer due to high
imidazole concentration.
13. Superose 6 maximum pressure is 1.5 MPa; recommended flow
rate is 0.4 mL/min.
Acknowledgments
This work is supported through a BBSRC Responsive Mode Grant
(BB/P000940/1) and a Wellcome Trust Investigator Grant
(210701/Z/18/Z). We would like to thank Kyle Powers, Flint
Stevenson-Jones, and Josh Bufton for critically reading the
manuscript.
References
1. Babu M, Vlasblom J, Pu S et al (2012) Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae. Nature
489:585–589
2. Hattab G, Warschawski DE, Moncoq K et al
(2015) Escherichia coli as host for membrane
protein structure determination: a global analysis. Sci Rep 5:12097
3. Miroux B, Walker JE (1996) Over-production
of proteins in Escherichia coli: mutant hosts that
allow synthesis of some membrane proteins
and globular proteins at high levels. J Mol
Biol 260:289–298
4. Wagner S, Klepsch MM, Schlegel S et al (2008)
Tuning Escherichia coli for membrane protein
overexpression. Proc Natl Acad Sci U S A
105:14371–14376
5. Studier FW (2005) Protein production by
auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
6. Zorman S, Botte M, Jiang Q et al (2015)
Advances and challenges of membrane-protein
complex production. Curr Opin Struct Biol
32:123–130
7. Li GW, Burkhardt D, Gross C et al (2014)
Quantifying absolute protein synthesis rates
reveals principles underlying allocation of cellular resources. Cell 157:624–635
8. Smolke CD, Keasling JD (2002) Effect of gene
location, mRNA secondary structures, and
RNase sites on expression of two genes in an
engineered operon. Biotechnol Bioeng
80:762–776
Production of Multi-subunit Membrane Protein Complexes
15