The phage-
derive
d expression, packaging, an
d processing (PEPP) system was use
d to target foreign proteins into the bacteriophage capsi
d to probe the intracapsi
d environment an
d the structure of package
d DNA. Small proteins with minimal requirements for activity were selecte
d, staphylococcal nuclease (SN) an
d green fluorescent protein (GFP). These proteins were targete
d into the T4 hea
d by means of IPIII (internal protein III) fusions or CTS (capsi
d targeting sequence) fusions. A
dditional evi
dence is provi
de
d that foreign proteins are targete
d into T4 by the N-terminal ten amino aci
d resi
due consensus CTS of IPIII i
dentifie
d in previous work. Fusion proteins were pro
duce
d within host bacteria by expression from plasmi
ds or by pro
duc tion from recombinant phage carrying the fusion genes. Package
d fusion proteins CTS
der=0 SRC=/images/
glyphs/BF6.GIF>IPIII
der=0 SRC=/images/
glyphs/BF6.GIF>SN, CTS
der=0 SRC=/images/
glyphs/BF6.GIF>IPIII
TSN, CTS
der=0 SRC=/images/
glyphs/BF6.GIF>IPIII
der=0 SRC=/images/
glyphs/BF6.GIF>GFP, CTS
der=0 SRC=/images/
glyphs/BF6.GIF>IPIII
TGFP, an
d CTS
der=0 SRC=/images/
glyphs/BF6.GIF>GFP, where
der=0 SRC=/images/
glyphs/BF6.GIF> in
dicates a linkage pepti
de sequence Leu(Ile)-N-Glu cleave
d by the T4 hea
d morphogenetic proteinase gp21
during hea
d maturation, are observe
d to exhibit intracapsi
d activity. SN activity within the hea
d is
demonstrate
d by loss of phage viability an
d by
digeste
d genomic DNA patterns visualize
d by gel electrophoresis when viable phage are incubate
d in Ca
2+. Green fluorescent phage result imme
diately after packaging GFP pro
duce
d at 30&
deg;C an
d below, an
d continue to give green fluorescence un
der 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are pro
duce
d in bacteria at 37&
deg;C, an
d phage package
d with these proteins achieve a fluorescent state after incubation for several months at 4&
deg;C. GFP-package
d phage an
d prohea
ds analyze
d by fluorescence spectroscopy show that the mature hea
d an
d the DNA-empty prohea
d package i
dentical numbers of GFP-fusion proteins. Encapsi
date
d GFP an
d SN can be injecte
d into bacteria an
d rapi
dly exhibit intracellular activity.
In vivo SN
digestion of encapsi
date
d DNA gives an intriguing pattern of DNA fragments by gel ana
lysis, pre
dominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome
digestion la
dder, This quasi-limit DNA
digestion pattern, reache
d >100-fol
d more slowly than the loss of titer, is invariant over a range ≤10 to 200 molecules of SN package
d per hea
d, an
d in
depen
dent of proteolytic cleavage of SN from the IPIII portion of the fusion, favoring a
discontinuous package
d DNA structure. Ro
ds of
B-form DNA coul
d be envisione
d as protecte
d from
digestion, whereas bent or kinke
d DNA woul
d be more susceptible to the
diffusible SN. Such
discontinuous package
d DNA structures are favore
d for phage T4 by a number of lines of evi
dence.