TABLE 1.

LF substrates isolated from second-iteration substrate phage library

Positions surrounding the scissile bond are numbered as P8 to P4′ from the N to C terminus of the substrate. The scissile bond is P1 to P1′. Substrates were selected as cleavable by LF after screening the second-iteration library. To prepare the second-iteration library, conserved amino acids identified from the eight-amino acid library screening were fixed, and other positions were either partially (P7, P6, P3, and P2) or fully (P1 to P4′) randomized. Values accompanying amino acid symbols reflect the frequency of respective amino acids (%) at the indicated positions calculated after analysis of 31 isolated LF substrates. Only amino acid residues with ≥4% frequency are shown. N, any nucleotide; V, C/T/G; W, A/T; Y, C/T; R, G/A; D, A/G/T; H, A/C/T; X, any amino acid. Several abundant amino acids (for example, F in p3) were not programmed and may have emerged by spontaneous point mutation. LF15 is a substrate that was previously isolated from a synthetic peptide library (7) and is cleaved efficiently by LF.

	Cleavage positions
	P8	P7	P6	P5	P4	P3	P2	P1	P1′	P2′	P3′	P4′
Sub-library DNA sequence	CGT	DWH	WYC	CGT	CGC	RTT	WHH	NNV	NNV	NNV	NNV	NNV
Sub-library amino acid sequence	R	K/N/I/E/D/V/Y/L/F	T/S/I/F	R	R	V/I	T/S/N/K/P/H/I	X	X	X	X	X
Frequency of amino acid residues, %	R(100)	I(60.46)	I(63.04)	R(100)	R(100)	V(82.61)	N(47.83)	S(47.83)	S(56.52)	L(51.16)	P(47.73)	L(63.04)
		D(11.63)	F(19.57)			I(13.04)	S(13.04)	A(10.87)	P(13.04)	R(6.98)	L(15.91)	G(6.52)
		Y(9.30)	R(8.70)			F(4.35)	I(10.87)	L(8.70)	T(6.52)	G(6.98)	I(6.82)	H(6.52)
		V(6.98)	T(4.35)				L(8.70)	P(6.52)	G(4.35)	S(6.98)	G(4.55)	R(6.52)
		E(4.65)	S(4.35)				T(8.70)	R(6.52)	Y(4.35)	K(4.65)	S(4.55)	E(4.35)
		N(4.65)						E(4.35)		I(4.65)		V(4.35)
								G(4.35)		P(4.65)
The resulting consensus	R	I	I	R	R	V	N	S	S	L	P	L
LF15		R	R	K	K	V	Y	P	Y	P	M	E