The development of safe and efficient gene delivery vectors with tailored properties is a fundamental goal of gene therapy. Non-viral gene delivery vectors have the potential to overcome immunogenicity and scale-up issues associated with viral vectors. However, the low transfection efficiency of many non-viral vectors has limited their use. To achieve sufficient transfection efficiencies, modular non-viral vectors containing functional groups to overcome extracellular and intracellular barriers to gene delivery are being developed based on studies of viral function and cellular processes. Specifically, the incorporation of nuclear localization signals (NLSs) into non-viral vectors has been employed to mediate trafficking of DNA complexes to the nucleus. We hypothesize that the presentation of the NLSs within the vector, which can be manipulated by using chemical conjugation and genetic engineering, may play a role in the accessibility and effectiveness of the NLSs. Using recombinant techniques, protein polymer vectors comprised of repeating peptide units were expressed in E. coli and purified using affinity chromatography, with their molecular weights verified by MALDI-TOF mass spectrometry. These protein polymers contain the minimal sequence of the SV40 nuclear localization signal, PKKKRKV, either repeated in the protein polymer backbone or conjugated to a cationic protein polymer at every seventh residue to produce a branched protein polymer. Results demonstrate the successful synthesis of the NLS peptide branch, EGPKKRKVG, by solid-phase synthesis. The peptide was purified by HPLC, achieving ~ 98% purity. The addition of this NLS peptide to Lipofectamine/DNA complexes resulted in a 17.5-fold increase in transfection efficiency of NIH3T3 cells. In addition, transfection efficiency using this peptide with Lipofectamine was comparable to lipofection without the peptide using more than twice the amount of DNA, suggesting that the inclusion of this peptide into DNA complexes may reduce the amount of DNA needed for in vitro transfection. These results indicate that combining bioconjugate chemistry and genetic engineering techniques can provide the tools for novel investigations of the mechanisms of non-viral gene delivery.