TABLE 2.

Single and double point mutations in the context of Mini-RAG1

	Position and mutationa	Amino acidb	Protein yieldc	RAG2 bindingd
1	524D	Lys	++++	+
2	531K	Asp	++++	++
3	541N	Leu	++++	+
4	546N	Asp	++++	−
5	547Q	Gln	++++	−
6	551K	Asp	++++	++
7	997Q	Lys	++++	−
8	997E	Lys	++++	+
9	1001E	Lys	++++	++
10	S517N/S519N	Phe-Trp	−	NDe
11	N529S/N530A	Arg-Thr	++++	+
12	Lys-546/Lys-547	Asp-Glu	++++	−
13	Asn-536/Gln-Q547	Asp-Glu	++++	−

^a All mutants were N-terminal MBP- and C-terminal His₆-tagged.

^b These are the residue(s) mutated.

^c Protein yield and quality were evaluated by FPLC. ++++, expression typical for MBP-Mini-RAG1, which is ∼5-fold greater than that of MBP-R1c; −, no protein expression detected.

^d The ability to bind to GST-RAG2 core was tested by BLItz. ++, binding as seen between R2c and either R1c or Mini-RAG1; +, binding reduced 3–20 fold; −, little or no binding detected.

^e ND means not done.