The monoclonal myth

Abstract

Most researchers confidently assume that transformation of recombinant plasmid libraries into microbial hosts followed by outgrowth of isolated colonies results in a "one cell-one mutant gene-one protein variant" paradigm. Indeed, this assumption is supported by the overwhelming majority of published studies employing bacterial expression hosts. In stark contrast, we recently reported on Saccharomyces cerevisiae libraries containing unexpectedly high frequencies of cells harboring heterogeneous mixtures of plasmids, so called Multiple Vector Transformants (MVT). Intriguingly, we observed that yeast MVT persist as a significant proportion of populations for multiple generations. MVT can lead to misidentification of isolated mutants loss of functionally enhanced clones, and unwitting propagation of false positives derived from contaminating control sequences. Such experimental complications can have devastating outcomes in the context of protein engineering by combinatorial library screening. Herein, we demonstrate that the phenomenon of MVT is not restricted to vectors bearing the CEN/ARSH origin of replication, but may be an even greater concern when using high copy 2 µm plasmids. To mitigate the risks associated with MVT, we have developed an optimized sequencing procedure that facilitates rapid and reliable identification of MVT among clones of interest. In our experience, MVT and their associated risks can be virtually eliminated by employing extended liquid outgrowths of transformed populations and archiving sequence-verified, monoclonal, mutant genes from cell-templated PCR amplicons.

Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein

“…Lysozyme variants were purified as described in detail elsewhere. 16 Protein Characterization. The lytic activities of lysozymes were analyzed by light scattering assays as previously described.…”

“…DNA, F-actin, mucin, and alginate). To address this limitation, hLYZ’s electrostatic potential field has been redesigned (16, 17), and the engineered variant has shown improved efficacy in a murine model of Pseudomonas aeruginosa lung infection (18, 19). More generally, this successful redesign of hLYZ has led us to conclude that putative limitations of the wild type protein can be addressed through molecular engineering of performance enhanced variants.…”

“…For example, in pulmonary infections, hLYZ’s cationic character is known to drive electrostatic mediated aggregation with and inhibition by negatively charged biopolymers that accumulate in the infected lung (e.g., DNA, F-actin, mucin, and alginate). To address this limitation, hLYZ’s electrostatic potential field has been redesigned, , and the engineered variant has shown improved efficacy in a murine model of Pseudomonas aeruginosa lung infection. , More generally, this successful redesign of hLYZ has led us to conclude that putative limitations of the wild type protein can be addressed through molecular engineering of performance enhanced variants.…”