Figure 4
From: One-step generation of error-prone PCR libraries using Gateway® technology
Representative results of library screening and sub-cloning experiments. (A) The fluorescence to OD600 ratio (mean value and standard deviation of a triplicate experiment) of the clones indicated on the × axis were determined as described in Methods. Z, Leucine zippers; S, Stop-NTAIL; N, wt NTAIL; 1-3, full length mutated NTAIL; 4, truncated NTAIL mutant. (B) His-tagged proteins expressed by clones Z, S, N, 1-4 (Figure 4A) were purified by affinity chromatography on IMAC as described in Methods, and were analyzed by SDS-PAGE using 15% polyacrylamide gels and Coomassie blue staining. Soluble, His-tagged proteins were purified under non denaturing conditions from the soluble fraction of the E. coli lysate. Total, His-tagged proteins were purified under denaturing conditions from total E. coli lysate. Soluble and total fractions were obtained from a duplicate culture. M, molecular size markers (from top to bottom: 170, 130, 100, 70, 55, 40, 35, 25, 15, 10 kDa). Arrows indicate the different purified proteins: 1, NGFP- wt NTAIL and NGFP-full-length NTAIL variants (34 kDa); 2, NGFP-truncated NTAIL variant 4 (29.4 kDa); 3, NGP-Z (22.8 kDa); 4, NGFP (i.e., Stop-NTAIL) (20.4 kDa); 5, XD-CGFP (15.5 kDa); 6, Z-CGFP (13.3 kDa). (C) PCR screening of mutated NTAIL sub-cloning experiment from pNGG to pDEST17O/I. PCR control a, 1,046 bp; b, 560 bp; c, 333 bp. PCR screening II and III were run in the same gel along with controls a, b, and c. M, molecular size markers (from top to bottom: 2000, 1500, 1000, 750, 500, 250 bp).