Soluble expression, purification and characterization of the full length IS2 Transposase

Lewis, Leslie A; Astatke, Mekbib; Umekubo, Peter T; Alvi, Shaheen; Saby, Robert; Afrose, Jehan

doi:10.1186/1759-8753-2-14

Mobile DNA

Table 2 In vitro electrophoretic mobility shift assays, binding efficiencies and in vivo LacZ papillation assay-determined transposition frequencies from IS2 wild type and mutant (GMF^{a, d}) isolates

From: Soluble expression, purification and characterization of the full length IS2 Transposase

Column number	1	2	3	4	5	6	7	8	9
Row number	Description of wild type and mutant GMF plasmids/strains.	Binding efficiency^a	Description or mutation	Domain-location of mutations^b	Total number of colonies	Number of trials (n)	Number of colonies with papillae	Total number of papillae	Transposition frequency^c
1	pUH2523ΔorfAB^c	-	Null	-	83	5	14	15	0.18 ± 0.16
2	pUH2509^d	-	WT OrfAB	-	96	2	66	137	1.25 ± 0.23
3	pLL2522^a/pUH2523^d	5.0	WT fusion protein	-	174	6	95	254	1.28 ± 0.09
4	pLL2524^a/pUH2524-004^d	5.0	A42T	H + HTH	43	3	24	64	1.31 ± 0.20
5	-009	3.0	R50H	H + HTH	18	1	2	2	0.00
6	-013	3.0	S57G	H + HTH	66	4	26	33	0.32 ± 0.17
7	-028	0.0	S44N	H + HTH	59	2	18	24	0.23 ± 0.08
8	-029	0.0	L58I	H + HTH	68	2	14	17	0.07 ± 0.07
9	-034	0.0	R13H	H + HTH	97	2	12	12	0.00
10	-036	2.0	R37Q/S44N	H + HTH	162	4	51	73	0.27 ± 0.11
11	-037	5.0	W49R	H + HTH	62	2	4	4	0.00
12	-040	4.0	V35L	H + HTH	52	3	38	75	1.26 ± 0.18
13	-006	2.5	Q79L	LZ-L	137	3	10	10	0.00
14	-007	3.0	N94D	LZ-L	52	2	14	16	0.13 ± 0.09
15	-018	3.5	A42T/L97H^e	LZ-L	44	2	13	17	0.21 ± 0.15
16	-094	ND	K89M	LZ-L	43	3	2	2	0.00
17	-106	0.0	L83V	LZ-L	133	4	4	4	0.00
18	-003	2.5	R291H	CAS	59	1	11	10	0.00
19	-022	2.0	A341P	CAS	88	5	16	18	0.09 ± 0.11
20	-024	0.5	L266P	CAS	34	2	4	4	0.00
21	-031	0.5	V301M	CAS	60	2	2	2	0.00
22	-038	5.0	A341T	CAS	37	2	30	80	1.98 ± 0.21
23	-068	2.5	H267D	CAS	106	3	17	19	0.00
24	-071	2.5	E391K	CAS	139	4	19	28	0.20 ± 0.26
25	-096	5.0	W237R	CAS	80	4	10	16	0.02 ± 0.05
26	-101	3.0	V179L	MI	113	4	7	10	0.00

^aBinding efficiencies were determined from electrophoretic mobility shift assays as described in the Methods section and illustrated in Figure 6. The wild type OrfAB-GFP fusion protein was expressed from pLL2522 and mutant OrfAB-GFP-GMF proteins from pLL2524-XXX plasmids (1-110). ^bDomains and locations of mutations are as described in Table 1. ^cTransposition frequencies were calculated as the number of papillae per colony (column 8 divided by column 5) minus the background frequency of transposition calculated from the null mutation (row 1). Frequencies shown for the mutants reflect only their contributions to the observed results (column 8/column 5). When 0.0 is listed, the observed result is less than the background frequency probably due to experimental error or variation in the count which may have been a function of sample size. The null mutation was derived from self ligation following an Mfe I digestion which removed most of the IS2orfAB::GFP fusion from the pUH2523 plasmid. The background frequency of transposition from the null mutant is likely due to the presence of IS2 copies on the chromosome of JM105 into which plasmids used in the LacZ assay were transformed (see Methods). ^dPlasmids used to determine transposition frequencies by means of the LacZ papillation assay were pUH2509 for the WT OrfAB protein, pUH2523 for the WT OrfAB-GFP protein and pUH2524-XXX (1-110) for the mutant OrfAB-GFP-GMF proteins. ^eThe phenotype of GMF 18 is attributed to the L97H substitution since both the binding efficiency and the transposition frequency of the A42T substitution (row 4) do not differ statistically from those of the wild type (row 3). CAS: putative catalytic active site; H + HTH: the binding domain; LZ-L: leucine zipper-like oligomerization motif; MI: the middle interval; WT: wild type.

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ISSN: 1759-8753

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