Anchoring Proteins To Escherichia Coli Cell Membranes Using Hydrophobic Anchors Derived From A Bacillus Subtilis Protein Mrpf

In this research, a Bacillus subtilis membrane protein MrpF was used as an anchor for anchoring hydrophilic proteins to the Escherichia coli cell inner membrane. And a common and stable hydrophilic protein PhoA was selected to be anchored. At first we used the computer to predict the structure and hydrophobicity of MrpF. And the result showed that MrpF was a three-time transmembrane protein. Based on computer prediction, the fusion protein PhoA-MrpF was designed. The phoA gene from E. coli was cloned, and then fused to upstream of the gene mrpF, then introduced into E. coli to express. The results showed that the fusion protein showed highly PhoA activity, and anchored to the membrane. This result proved the anchor MrpF in the cell membrane did not significantly affect the expression and activity of PhoA.Then, MrpF was the truncated. When MrpF was truncated to only one transmembrane helix domain, PhoA still showed high activity, and still anchored in the cell membrane. Further study was performed on truncated MrpF to find the shortest anchor. The cell PhoA assay was developed. One was lysozyme-EDTA treatment。 The cell wall was digested by lysozyme-EDTA so that the periplasmic PhoA can directly exposed, free PhoA will be washed and anchored PhoA was still in the membrane. Another method is osmotic shock. This method can release periplasmic proteins of E.coli from the periplasm, but if PhoA is anchored in the membrane, it will not be released. On the basis of computer prediction, a series truncated MrpF and alkaline phosphatase fusion proteins were builded, and then introduced into Escherichia coli to express. Using the method mentioned above to assay the efficiency of anchoring. The length of hydrophobic anchors was found to be critical for anchoring proteins to cell membranes. And the shortest length for anchoring was8amino acid residues.The conclusions can be described from theory and applications. First, the machinism of how the protein transmembrane transporting, and how the integral membrane proteins inserting into the lipid membrane, is still not clearly. In our study, we find a shortest hydrophobic tag length for membrane anchoring. And this length may be the shortest length for stop transferring which recognized by translocon. Second, in the APEx system, the protein designer may benefit from this conclusion when desining their protein.