A phasmid optimised for protein design projects: pMAMPF

Abstract

Protein design requires the rapid production of recombinant genes and active recombinant proteins, the latter in sufficient amounts for functional and physical studies. We present here the construction and application of a new phasmid vector system, using the fd phage origin, Λp L prometer, ompA-leader sequence and pMB1 origin, which allows the preparation of secretable proteins in active form, mutagenesis and gene sequncing, without subcloning steps. The vector can be used in plasmid form in a stably transformed culture to induce product formation, or as a packaged single-standed phasmid, which via batch transduction in a growing culture, leads directly to recombinant protein formation. This latter method has the advantage that, during the short period required for phasmid amplification, little counterselection against clones with high rDNA-protein synthesis potential occurs. The total sequence of pMAMPF-1 and pMAMPF-3 can be assembled from known sequences of constituent fragments. Mutated regions were directly sequenced.