The Sreening And Characterization Of TAT-PTD Interacting Proteins From Cell Membrane Based On TAP Technique

The trans-activator of transcription（TAT）,derived from human immunodeficiency virus-1 （HIV-1）,is the most researched cell penetration peptide（CPP）.It is powerful at penetrating cell membrane and can transport the conjugated cargos into cells without affecting their biological activity,so,it has become the hot spot of the development of biological medicine and the cytological research.Researchers have found that,a peptide rich in basic amino acids, YGRKKRRQRRR,called protein transduction domain（PTD）,is responsible for the proteintransporting function of HIV-TAT.It is the minimum structure that has the function of transporting proteins and it can transport proteins efficiently.Early research on the transmembrane transduction mechanism of TAT showed that,TAT-PTD followed a dynamic transmembrane transportation mechanism to enter cells and it was independent of temperature,energy and receptor.It was speculated that the basic amino acid residues（arginines and lysines） might play an important role in this mechanism and TAT’s ability of penetrating cell membrane depended largely on these basic amino acid residues:these electropositive amino acid residues could interact directly with the electronegative cell membrane lipid and then penetrated cell membrane with the help of electrostatic interaction.But,later research found that TAT could interact directly with heparan sulfate proteoglycan（HSPG） to initiate the protein transduction process because that TAT carried a lot of positive charge while HSPG was electronegative.Heparin sulfate（HS）,a kind of glycosaminoglycan（GAG）,might be the key regulator of protein internalization.In the HS-deficient cells,the transduction function of TAT is inhibited significantly.Besides,addition of soluble heparin,HS simulator or enzymes that degrade HS to the cell culturing medium can also inhibit the internalization of TAT-conjugated proteins.During metabolism,various kinds of materials go into and out of cells constantly.Except for the small molecular substances,macromolecular substances and some granular materials enter cells through internalization,internalization is involved in multiple physiological and pathological processes.Endocytosis is an important way of internalization and it is a very complicated biological process.Now,there are two main ways of internalization for macromolecular substances like protein and peptide:（1） the receptor-mediated clathrin-dependent internalization way,which is utilized by most biomacromolecules that have been detected;（2） the cell membrane-mediated non-classical internalization way,which participates in the internalization of many kinds of bacteria and virus.HSPG,a kind of glycoprotein,exists widely on the surface of almost all the mammalian adherent cells,and it can also be found in the extracellular matrix and basement membrane.HSPG is a complex macromolecule comprised of one core protein molecule and one or more glycosaminoglycan,and it plays an important role in cell adhesion,proliferation, differentiation,vascular integrity and vascular permeability.Tandem affinity purification（TAP） is a technique established by Rigaut et al in 1999.At first, it was used to express fusion proteins with affinity tags in cells using molecular cloning technique, and then,the bait protein was separated and purified from prokaryotic cells though the interaction of affinity purification tags with corresponding magnetic beads.Now,TAP is widely used in the research of many species including mammals,yeast,bacteria,fruit fly and plants.The TAP bait protein complex would be repeatedly purified with two or more purification tags,and after two steps of combining and eluting,we could get the highly-purified protein complex with natural conformation.So,this technique could be applied to study protein-protein interactions under physiological environment.The highly-purified proteins,which truly interact with the target protein,could be easily obtained through the interaction of the specified tags with affinity columns.Recently,Stratagene company has developed the InterPlay Mammalian TAP System, which is suitable for mammalian cells and can be applied to detect interacting proteins in these cells.The refined system substitutes Prot A and tobacco etch virus（TEV） with the purification tag streptavidin-binding peptide（SBP） and this modification avoids the influence of TEV protease on the target protein and minimizes the contamination of macromolecular substances,making the separation process much easier.The bait protein complex was purified twice with two purification tags:cahnodulin binding peptide（CBP） and SBP,and then it was eluted with two mild elution steps（EGTA and biotin）.In the end,the highly-purified protein complex with natural conformation can be obtained.It is a significant breakthrough in the methodology of protein-protein interactions and it provides a new method for studying the protein-protein interaction network and proteomics in higher eukaryotes.The combination of TAP with mass spectrometry makes it possible for us to analyze protein-protein interactions on a large scale.Understanding the protein-protein interaction network is very important for us to precisely understand the function of proteins and unveil some intricate life secretes.Based on the understandings mentioned above,in our experiment,we constructed several enhanced green fluorescent protein（EGFP） fusion protein expression vectors with His,CBP and SBP purification tags,including His-CBP-SBP-EGFP,His-CBP-SBP-EGFP-TAT,His-CBP-EGFP, His-CBP-EGFP-TAT,His-SBP-EGFP,and His-SBP-EGFP-TAT.After the induction of expression in BL21（DE₃） host bacteria and the purification with Ni²⁺-NTA,we got the corresponding fusion proteins.Then,we studied the transmembrane transduction function of these fusion proteins with the live cell image analyzing system.Based on the fundamental principle of TAP,we successfully established the TAP pull-down technique.Heparin,a simulator of heparan sulfate,was used to select the mouse liver membrane proteins that interact with TAT-PTD. Through the identification with mass spectrometry,we found some proteins that interacted with TAT-PTD,and that is very meaningful for elucidating its membrane-penetrating mechanism.Through our research,we have achieved some conclusions as follows:A.We have successfully constructed two prokaryotic expressing fusion protein expression vectors:His-CBP-SBP-EGFP and His-CBP-SBP-EGFP-TAT.We confirmed that the His-CBP-SBP -EGFP-TAT could be efficiently transducted into HepG2 cells,and the transduction effect is dependent of time and concentration.B.We found that the control His-CBP-SBP-EGFP could attach to cell membrane by chance. In order to explain this phenomenon,we constructed the CBP or SBP deficient His-CBP-EGFP, His-CBP-EGFP-TAT,His-SBP-EGFP and His-SBP-EGFP-TAT vectors on the basis of the His-CBP -SBP-EGFP and His-CBP-SBP-EGFP-TAT vectors.By comparing the transmembrane effect of these fusion proteins on the cell level,we found that the SBP-deficient His-CBP-EGFP fusion protein could attach to cell membrane while the CBP-deficient His-CBP-EGFP could not attach to cell membrane.This finding showed that the CBP tag of TAP could change the localization of fusion proteins in cells,and it might be caused by the interaction of CBP with some structural component on the cell membrane.C.In order to avoid the nonspecific protein background added by the CBP tag during purification,we used the His-SBP tandem affinity purification tag as the purification system for TAT-PTD interacting proteins.The SDS-PAGE analysis revealed that,after the second pull-down with SBP,the background of the TAT-PTD interacting proteins,which were initially obtained by His pull-down,dropped dramatically.There were less nonspecific proteins and an obvious differential protein band was revealed.This protein band was identified with mass spectrometry and was confirmed to be complement component 1,q subcomponent binding protein（C1qbp） with 100%credibility.C1qbp exists mainly in the matrix and membrane of mitochondria,and it might play an important role in the transmembrane transportation of macromolecular substances.In summary,based on the principle of TAP technique,we have successfully constructed the prokaryotic expressing EGFP fusion protein expression vector containing the His-CBP-SBP tandem purification tag.We also optimized this system and affirmed that the CBP-deficient His-SBP tandem purification tag had higher specificity and could reduce the background significantly. Therefore,it was proven to be an important tool for studying the transmembrane mechanism of TAT-PTD and its localization in cells.The mouse liver membrane protein was prepared and then the protein complex was purified by the TAP pull-down experiment with the His-SBP tandem affinity purification tag.After the two purification steps with His and SBP,we obtained the TAT-PTD interacting protein with high specificity and natural conformation.The differential protein was identified as C1qbp by the mass spectrometry analysis,providing a new perspective for the study of the transmembrane transduction mechanism of TAT.