Gene cloning, purification, and stoichiometry quantification of phi29 anti-receptor gp12 with potential use as special ligand for gene delivery.
Bacterial virus phi29 is the most efficient in vitro DNA packaging system, with which up to 90% of the added DNA can be packaged into purified recombinant procapsid in vitro. The findings that phi29 virions can be assembled with the exclusive use of cloned gene products have bred a thought that phi29 has a potential to be a gene delivery vector since it is a nonpathogenic virus. gp12 of bacterial virus phi29 has been reported to be the anti-receptor that is responsible for binding the virus particle to the host cell. We cloned the gene coding gp12, overexpressed it in Escherichia coli, and purified the gene product to study the properties and functions of gp12 in virus assembly. According to SDS PloyAcrylamide Gel Electrophoresis (SDS-PAGE) analysis and N-terminal sequencing, recombinant gp12 isolated from E. coli had a molecular mass of 80 kDa, and 24 amino acids at N-terminal were cleaved after expression. The purified recombinant gp12 was incorporated into phi29 particles and converted the gp12-lacking assembly intermediates of phi29 into infectious virions in vitro. This purified protein gp12 was able to compete with infectious phi29 virions for binding to the host cell, thus inhibiting the infection by phi29. Scanning Transmission Electron Microscopy (STEM) analysis and sedimentation studies revealed that recombinant gp12 products were assembled into biologically active dimers. Analysis of the dose-response curve showed that 12 dimeric gp12 complexes were assembled onto viral particles and that each virion contained 24 copies of gp12 molecules. The results provide a basis for future research into bacteriophage-host interaction by modifying the anti-receptor protein. The ultimate goal is to re-target the bacteriophage to new host cells for the purpose of gene delivery.
Guo
Songchuan
S
Laboratory of Molecular Virology and Gene Therapy, Purdue University, West Lafayette, IN 47907, USA.
Shu
Dan
D
Simon
Martha N
MN
Guo
Peixuan
P
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D018014:Gene Transfer Techniques; D008024:Ligands; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014771:Virion / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D019065:Virus Assembly / Q000187:drug effects
D004279:DNA, Viral; D008024:Ligands; D011994:Recombinant Proteins; D014764:Viral Proteins