Table III.

Comparison of features of preparation methods

Reference	This study		Kigawa et al. (25)	Kwon and Jewett (45)	Fujiwara and Doi (67)
Strain	BL21/pMINOR2		BL21-Codon Plus-RIL	BL21 StarTM (DE3)	BL21-Codon Plus (DE3)-RIL
Culture vol. for a batch	500 ml–60 l		2–8 l	10 ml–10 l	1–9 l
Cell conc. at disruption	Variable from 1 to 0.04 g/ml		Fixed at 7 g per 8.9 ml	Optimized at 1 g/ml	Optimized at 0.67 g/ml
Cell disruption	High pressure cell homogenizer		Multi-beads shocker	Q125 Sonicator	LoFT*a
Centrifugation	30k × g, 30 min		30k × g, 30 min	12k × g, 30 min	25k × g, 60 min
Pre-incubation	37 °C, 80 min		37 °C, 80 min with chemicals*b	37 °C, 60 min	–
Removal of low-molecular chemicals	Diafiltration by TFF system*c		Dialysis	–	Diafiltration*d
Adjustment of conc. of extract	Based on A260/ml		–	–	Based on volume
Protein yield*e	GFPS2	CAT	CAT	sfGFP	sfGFP
batch mode (mg/ml)	–	–	0.8 (37 °C, 1 h)	0.5 (37 °C, 4 h) 1.0 (20 h)	0.5 (29 °C, 14 h)
dialysis mode (mg/ml)	1.1 (25 °C, 4h)	6.6 (30 °C, 16 h)	5.4 (30 °C, 16 h)*f	–	–

^aLysozyme treatment, osmotic shock and freeze-thawing method.

^bAll chemicals are listed in Supplementary Table SI.

^cCentrifugal ultrafiltration was used for <100-ml extract.

^d1 ml of extract was diluted by 13 ml of buffer and concentrated by centrifugal ultrafiltration to be 1 ml.

^eYields are shown in mg of protein per reaction solution (mg/ml) and the reaction temperature and time are shown in parentheses.

^fThe yield of dialysis mode was reported by Seki et al. (53).