Identification of Novel Macropinocytosing Human Antibodies by Phage Display and High-Content Analysis.

Internalizing antibodies have great potential for the development of targeted therapeutics. Antibodies that internalize via the macropinocytosis pathway are particularly promising since macropinocytosis is capable of mediating rapid, bulk uptake and is selectively upregulated in many cancers. We hereby describe a method for identifying antibodies that internalize via macropinocytosis by screening phage-displayed single-chain antibody selection outputs with an automated fluorescent microscopy-based high-content analysis platform. Furthermore, this method can be similarly applied to other endocytic pathways if other fluorescent, pathway-specific, soluble markers are available.

Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides.

Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.