One-step purification is enabled by the hexahistidine affinity tag and metal affinity chromatography (IMAC) and performed under denaturing conditions.
A fully automated protein purification system has been developed to allow for purifications of up to 60 cell lysates at a time. coli shake flask cultures upon induction with IPTG. Plasmids are collected from all purified clones for deposition in the clone library and glycerol stocks are prepared and used as starting material for protein production.Īll proteins are expressed as His6ABP fusions in E. coli Rosetta(DE3), inserts are verified by DNA sequencing to omit clones with mutations and approved clones are single cell streaked. (2000)) where the human gene fragment is fused to a histidine tag and albumin binding protein (His6ABP). Amplicons are automatically processed with solid phase restriction, and ligated into the plasmid vector pAff8c ( Larsson M et al. (2005)) are first amplified with RT-PCR from total RNA template pools with specific oligonucleotide primers for each PrEST.
0 kommentar(er)
