In Vitro And In Vivo Analysis Of The TAT-mediated Protein Transduction As Cellular Delivery Vehicle

ObjectiveMost of the biopharmaceutical drugs, such as peptides. proteins, enzymes and oligonucleotides, have very limited effectiveness in clinical applications since they lack the ability to penetrate into cells and reach their pharmacological targets. Extensive studies have been conducted in an attempt to develop suitable delivery systems which can carry biopharmaceutical drugs across the impermeable plasma membrane for therapeutic purpose. HIV-1 transactivator of transcription (TAT) has demonstrated ability to traverse the cellular membrane in many studies. Recently, we conducted a systematic study to explore the mechanism of the TAT-mediated protein transduction process.Giant molecular substance go straight through cellular membrane difficultly as the selective permeability of the cellular membrane. So the cellular membrane becomes the main reason of the giant molecular drug producing a marked effect to human body. Many kinds of protein,polypeptide and oligonucleotide that has the effect of curing to human body go through the cellular hardly to produce marked effect. We are due to reserch protein transductive domain that can take along giant molecular drug to go through the cellular membrane.MethodsWe constructed an expression vector pET-28a-TAT-EGFP and expressed a quantity of His-TAT-EGFP fusion protein in E. coli. cells, purified the fusion protein using affinity chromatography. The fusion protein His-TAT-EGFP was confirmed with DNA sequence and protein sequence. With the same procedure, we also obtained His-EGFP as the negative control. The fusion peptide His-TAT-EGFP was incubated with HeLa cells and PC 12 cells at 37℃temperature for 60 minutes. After rinsed three times. these cells were examined under fluorescence microscope. The fluorescence microscopic images showed that the fusion protein His-TAT-EGFP is able to traverse the cellular membrane in 20 minutes, whereas the protein His-EGFP cannot penetrate into the cells. In vivo experiments were also conducted where fusion protein His-TAT-EGFP and His-EGFP were injected intraperitoneally into Kunming mice, respectively. Two hours later, these mice were sacrificed and their organs (liver, brain, heart and kidney) were obtained and made frozen slides. All the slides showed fluorescent His-TAT-EGFP under the fluorescence microscope, of these mice which were injected with protein His-EGFP did not show any observable fluorescence signals. The data also showed that His-TAT-EGFP can reach and penetrate into these organs within 2 hours. However, the data showed that TAT-mediated protein transduction did not show any tissue specificity.Through plasmid construction we construct plasmid that conect protein trunsductive domain and enhance green fluorescent protein gene order. Then through BL21 bacterium we expesses fusion protein and purificate fusion protein. Through fluorescent microscope overview to test the effect of going through cellular membrane in vitro and the effects of the protein going through the cellular membrane of live animal in vivo. Furthermore, we investigated the effect of the orientation and position of the TAT peptide on the protein transduction capability. The amino acid sequence of TAT was reversed (named as TAT(-)), and then fused with His-EGFP protein. The TAT and TAT(-) positions relevant to the His-EGFP protein were also changed. Thus, four fusion proteins were obtained, that is His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-), respectively. Comparison of the results did not exhibit significant difference in terms of the transducing capability. The current study provides a reliable experimental foundation on which we established a technical platform for constructing more flexible fusion proteins for therapeutical applications.ResultsResult displays:the four fusion proteins His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-) of expression have the effect of going through cellular membrane in vivo and in vitro, while His-EGFP doesn't have.ConclusionsThe plasmid construction can express the proteins His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-), which named protein trunsductive domain can take along giant molecular protein to go through cellular membrane in vivo an in vitro. It shows that different fusion protein has different transmembrane capacity, the TAT(-) sequence and in C terminal has stronger transmembrane capacity. It constructs a technique flat-top for exploitation new drug...