Development of a Novel Type of Cloning Vector for Suicide Selection of Recombinants

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Abstract
Based on the primary structure of the rat corticostatin R4 (antimicrobial defensin RatNP-1), a synthetic gene was designed, synthesized, and inserted into the IPTG-inducible prokaryotic expression vector pFLAG, with an additional 13 codons between the FLAG sequence and the synthetic R4 sequence. This construct, N-p1.2, was further developed by inclusion of multiple cloning sites right after the FLAG sequence, forming a new plasmid pSCV-1. Escherichia coli transformants containing pSCV-1 or N-p1.2 could only be propagated on agar plates in the absence of IPTG due to the detrimental expression of R4 fusion peptide to the growth of bacteria upon IPTG induction. A 214-bp bovine IGF-II cDNA and a 700-bp Ly-6C.2 cDNA fragment were subcloned into pSCV-1 and N-p1.2 respectively. Only the E. coli cells transformed with recombinant plasmids grew on IPTG agar plates. This "suicide" selection against nonrecombinants was further tested in cDNA library construction using pSCV-1. Analysis of plasmid DNA prepared from randomly picked colonies growing on ampicillin agar plates containing IPTG showed all plasmids contained cDNA inserts. The λHind HI fragments were used for comparing the cloning efficiency of pSCV-1 to pBluescript. Four of the 60 (6.6%) analyzed white colonies transformed with pBluescript were false positives. All of the analyzed pSCV-1-transformed colonies growing on IPTG plates contained recombinant forms of plasmid. The percentage recovery of each ligatable λHind III fragment was similar in both pBluescript and pSCV-1. After insertion of a sequence encoding bovine IGF-II so that an IGF-II/R4 fusion protein would be generated, bacteria containing this construct were able to grow and produce the fusion protein in the presence of IPTG, implying the 8-kD peptide fused to the amino-terminal end of R4 was sufficient to mask its antimicrobial activity.