Reliable scale-up of membrane protein over-expression by bacterial auto-induction: from microwell plates to pilot scale fermentations.
The production of well-ordered crystals of membrane proteins for structural investigation by X-ray diffraction typically requires extensive crystallization trials and may involve the screening of multiple detergents, lipids and other additives. Purification of sufficient amounts of protein for such trials is hampered by the fact that even when over-expressed, membrane proteins represent only a small percentage of the total protein content of bacteria. Fermentation-scale cultures of cells are therefore usually required. To maximize the efficiency and reduce the cost of such cultures, in the UK Membrane Protein Structure Initiative we have systematically investigated the use of auto-induction as an alternative to induction of expression with isopropyl-beta-D-thiogalactoside. We report here the benefits of first optimizing expression on a multiwell plate scale by systematically varying the concentrations of glucose, glycerol, lactose and succinate present in the auto-induction medium. For subsequent scale-up, comparison of isopropyl-beta-D-thiogalactoside induction in shake-flasks with auto-induction in shake-flasks and in 1L fermenters without and with control of pH and aeration revealed that highest yields of target protein were obtained using the latter culture conditions. However, analysis of the time-course of expression highlighted the importance of choosing the correct time for harvest. The high yields of target protein that can be obtained in a single batch by auto-induction, performed on a 30 l scale in a fermenter, obviate batch-to-batch variations that can add an unwanted variable to crystallization screening experiments. The approach described should therefore be of great utility for membrane protein production for structural studies.
Deacon
Sarah E
SE
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
Roach
Peter C J
PC
Postis
Vincent L G
VL
Wright
Gareth S A
GS
Xia
Xiaobing
X
Phillips
Simon E V
SE
Knox
J Paul
JP
Henderson
Peter J F
PJ
McPherson
Michael J
MJ
Baldwin
Stephen A
SA
D019149:Bioreactors; D018360:Crystallography, X-Ray; D003470:Culture Media; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis*; D010100:Oxygen / Q000378:metabolism; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000096:biosynthesis; D015533:Transcriptional Activation
D003470:Culture Media; D008565:Membrane Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D005947:Glucose; D007785:Lactose; D005990:Glycerol; D010100:Oxygen