Complexes of recombinant silk-polylysine molecules with ppTG1 peptide, a lysine-rich cell membrane destabilizing peptide to bind plasmid DNA (pDNA), are designed as less-cytotoxic and highly efficient gene carriers. The peptide destabilizes the cell membrane and promotes gene transfer. Our particular interest is in how ppTG1 enhances transfection efficiency of the silk-based delivery system into human cells. Genetically engineered silk proteins containing polylysine and the monomeric and dimeric ppTG1 sequences are synthesized in Escherichia coli , followed by transfection experiments. The pDNA complexes of silk-polylysine-ppTG1 dimer recombinant proteins prepared at an N/P 2 (the ratio of number of amines/phosphates from pDNA) shows the highest transfection efficiency into human embryonic kidney (HEK) cells, the level of which is comparable to the transfection reagent Lipofectamine 2000. The assemblies show a globular morphology with an average hydrodynamic diameter of 99 nm and almost no β-sheet structure. Additionally, the silk-based pDNA complexes demonstrate excellent DNase resistance as well as efficient release of the pDNA by enzymes that degrade silk proteins. Also, comparison with β-sheet induced silk-based pDNA complexes indicates that the β-sheet structure content of the silk sequence of the pDNA complexes controls the enzymatic degradation rate of the complexes and, hence, can regulate the release profile of genes from the complexes. The bioengineered silk-based gene delivery vehicles containing cell membrane destabilizing peptides are therefore concluded to have potential for a less-toxic and controlled-release gene delivery system.