Table 1. Mutations observed in evolved sortase variants.

Mutations in representative clones from rounds 1–16 are shown relative to the evolutionary starting sequence, 4S.6. These clones were the most abundant sequences in the evolving pool at the end of their rounds, with the exceptions of clone 8.5-H3 and SrtAβ, which were identified by single clone FACS at the end of round 8 and round 16, respectively.

4S.6	R1	R2	R4	R5	R7	R8	8.5-H3	R9	R10	R11	R12	R13	SrtAβ
K62						R	R	R	R	R	R	R
A73									V		V
I76						L		L	L	L	L	L	L
R94	P	Y	Y	Y	Y
S102													C
E105												D	D
N107							D	D	D	D	D	D	D
S118	I	I	I	I	I	I	I	I	I	I	I	I	I
A122		W
I123				L	L	L	L	L	L	L	L	L	L
D124		G	L	L	L	L	L	L	L	L	L	L	L
N127						Y			Y	H	H	H	H
G134	R	R	R	R	R	R	R	R	R	R	R	R	R
K138			I	I	I	L	I	I	L	L	L	L	L
G139													D
M141													I
K145						T			T	T	T	T	T
G147												C
N148											S
K152													R
M155										I	I	I	I
S157								R
R159						C		H	C	C	C	C	C
K162													R
D170										E	E	E
Q172									H	H	H	H	H
K173					E		E	E	E	E	E	E	E
K177							R	R					R
V182			A	A	A	A	A	A	A	A	A	A	A
V189	F	F	F	F	F	F	I	I	F	F	F	F	Y
T196			S	S	S	S	S	S	S	S	S	S	S
R197			S	S	S	S	S	S	S	S	S	S	S
K206									E	E			R