1853 2031

Expression of yFlp at 18 oC degree

Transform plasmid with BL21-AI/pRare2LysS. Induce with 0.1% arabinose and 0.15 mM IPTG, 18 oC ~18 hours.

Spin down 5 ml, lyse, pulldown using Ni beads.

	Clone ID	Plasmid Name
1	1870	pET28b-HTNFlp-ecoli 4oc
2	1870	pET28b-HTNFlp-ecoli 18oc
3	1853	pET28b-yFLP 4oc
4	1853	pET28b-yFLP 18oc
5	Fementas PageRuler Plus
6 &7

Use chaperones to improve Flp expression

1/25/2014

Test chaperone on Tat-yFLP expression

Lane	Name	Protein Name	Clone ID	Host	Induction	Purification	pI/Mw
1	pET28b-yFLP	Tat-yFLP	1853	5 ml, Tuner(DE3)-pG-KJE8	0.5 mM IPTG  RT o/n	Ni beads	9.76 / 52.8
2	pET28b-yFLP	Tat-yFLP	1853	5 ml, Tuner(DE3)-pG-KJE7	0.5 mM IPTG RT o/n	Ni beads	9.76 / 52.8
3	pET28b-yFLP	Tat-yFLP	1853	5 ml, Tuner(DE3)-pTf16	0.5 mM IPTG RT o/n	Ni beads	9.76 / 52.8
4	pCold-Tat-yFLP	Tat-yFLP	2031	5 ml, Tuner(DE3)	0.5 mM IPTG RT o/n	Ni  beads	9.76 / 52.8
5	Fermentas PageRuler Plus
6	pCold-Tat-yFLP	Tat-yFLP	2031	5 ml, Tuner(DE3)-pG-KJE8	0.5 mM IPTG RT o/n	Ni beads	9.76 / 52.8
7	pCold-Tat-yFLP	Tat-yFLP	2031	5 ml, Tuner(DE3)-pG-KJE7	0.5 mM IPTG RT o/n	Ni beads	9.76 / 52.8
8	pCold-Tat-yFLP	Tat-yFLP	2031	5 ml, Tuner(DE3)-pTf2	0.5 mM IPTG RT o/n	Ni beads	9.76 / 52.8
9	pCold-Tat-yFLP	Tat-yFLP	2031	5 ml, Tuner(DE3)-pTf16	0.5 mM IPTG RT o/n	Ni beads	9.76 / 52.8
10	pET28b-yFLP	Tat-yFlp	1853	5 ml, Tuner(DE3)-pTf2	0.5mM IPTG RT o/n	Ni beads	9.76 / 52.8
11	pET28b-yFLP	Tat-yFlp	1853	BL21-AI	0.5mM IPTG + 0.1%arabinose RT o/n	Ni beads	9.76 / 52.8

Conclusion: Chaperones do not enhance protein expression. Protein is expressed as truncated. Less than 53 kDa

Expression in pT7A3 and pCold

Other Protein Expression Screening