Fig.2.

Donor–Acceptor recombineering exploits. (A) Expression of the signal receptor particle SRP in E. coli was achieved by ACEMBL. A dual expression plasmid encoding for the protein subunit Ffh and the RNA subunit Ffs was created by fusing Donor and Acceptor plasmids containing the encoding genes. Co-expression resulted in SRP which eluted in a symmetric peak from a size-exclusion column. SDS–PAGE (Ffh) and agarose gel (Ffs) analysis of SEC fractions revealed the complex. SRP is depicted as a model (based on PDB entry 2IY3) in the inset. (B) E. coli was used as expression host to produce the SecYEG-DF-YidC holotranslocon. A schematic drawing of this transmembrane multiprotein complex bound to the ribosome is shown (top). A multigene ACEMBL construct was used to express and purify the holotranslocon (below). A gel section from SDS–PAGE of the complex purified from detergent solubilized membrane fractions shows all expected components (right). (C) Efficient multigene expression in mammalian cells. Five fluorescent marker proteins were inserted into Donors and an Acceptor containing mammalian cell active promoter elements. All cells produced all proteins at identical levels in all cells of the mammalian culture. EBFP2-Nuc stains the nuclei, mTFP-FYVE binds a phosphoinositol-phosphate (PI-3-P), tubulin is labeled with EYFP, Mito-dsRed localizes to mitochondria, and Plum-PLCδ-PH binds a phospho-inositol-biphosphate (PI-4,5-P2) The porcine aortic endothelial cells analyzed are shown in light field (bottom right). A and B are adapted from Bieniossek et al. (2009), C is adapted from a time-lapse movie in Kriz et al., 2010 (image credit to P. Berger, PSI Villigen, Switzerland).