Table 2.

Active site library selectants

Clone	Residues randomized					Increased display	Increased activity
Library 1	I31	D32	S3	G34
1.1 (17/4)	L	.	T	.		11	1.0
Library 2	H64	G65	T66	H67	V68
2.1 (5/1)	.	.	.	Y	.	1.5	0.13
2.2 (4/3)	.	.	.	A	.	3.2 ± 0.2	0.13 ± 0.003
2.3	.	.	.	*	.	2.1	0.065
Library 3	M124	S125	L126	G127
3.1	L	.	G	.
3.2	L	.	V	.		1.1	2.3
3.3	L	A	.	.		0.53	2.3
3.4	L	G	A	.		0.69	0.41
3.5	L	G	C	.		2.9	1.2
3.6	.	G	C	.		3.0	1.0
3.7	I	A	V	.		2.7	0.48
3.8	I	.	.	.
Library 4	A152	A153	G154	N155
4.1 (14/1)	C	.	.	.		3.1	0.068
4.2 (2/1)	S	.	.	.		0.40	0.079
Library 5	Y217	N218	G219	T220
5.1	.	S	.	.		2.6	0.63
5.2	A	S	.	.		3.4	0.72
5.3	C	T	.	.		3.4	1.9
5.4	V	T	.	.		1.7	0.58
5.5	M	A	.	.		2.7	0.19
5.6	G	H	.	.		2.2	0.61
Library 6	M222	A223	S224	A225
6.1 (2/2)	G	.	.	.		0.49	1.9
6.2	S	.	.	.		0.74	1.9
6.3	S	.	A	.		0.49	1.7
6.4	A	.	.	G		0.46	1.4
6.5	V	.	.	.		0.29	0.19
6.6	*	.	.	.		1.5	0.14
6.7	F	.	.	.		0.17	2.7
6.8	.	S	C	.		0.31	0.79

Mutations identified in subtiligase (see Fig. 4) after random mutation of six regions and sorting for four to seven rounds under product capture conditions as in Table 1. Periods indicate conservation of parent subtiligase residues. Asterisks indicate amber stops, suppressed as Gln in XL1-blue cells. Numbers in parentheses indicate the number of instances found for a particular amino acid sequence followed by the number of different DNA sequences. The improved activity on phage is normalized for improved display (see Materials and Methods). Assays on clones with the same amino acid sequence indicate that the results are reproducible to ±15%.