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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Protein Expr Purif. 2015 Dec 29;121:17–21. doi: 10.1016/j.pep.2015.12.001

Figure 4.

Figure 4

Anti-HIS Western analysis of wild-type and engineered cryptic site DHFR variant protein expressed in E. coli. (a) C-terminally HIS-tagged DHFR in crude extracts (lanes 1, 3) or metal affinity purified (lanes, 2, 4) complexes coexpressed in BL21(DE3)pLysS was detected using anti-DHFR antibodies. The wild-type DHFR sequence was used in lanes 1 & 2, while the DHFR sequence mutated to remove the cryptic Shine-Dalgarno mutation or START codon or both elements are shown in lanes 3 & 4 respectively. A polypeptide corresponding to translation from the engineered cryptic translation initiation site was not detected (b) Amino acid and DNA sequences for the wild type and cryptic initiation site DFHR mutant.