The Effect Of Signal Peptides On The Localization Of Membrane Protein

The membrane proteins expression and localization is one of the most challenge subjects for scientists. In this dissertation, effects of signal peptides on membrane protein expression and localization were explored. A small membrane protein, MrpF, a subunit of Mrp Na+/H+antiport from Bacillus subtilis was used as a model membrane protein in this investigation. Intracellular or periplasmic proteins are expressed and transferred across cytoplasmic membranes by signal peptides. A signal peptide consists of three parts as follows, the middle of the hydrophobic core area (h area), the C-terminal polar area (c area) and the N-terminus polar area (n area). In E. coli, two pathways, Sec and Tat, are responsible for proteins translocation and secretion. Here, three types of signal peptides were selected according to their involvement in protein translocations pathway, signal peptides of MalE (S1) and PhoA (S2) which are involved in Sec pathway, the signal peptide of TorA (S3) which is involved in Tat pathway, and the signal peptide of FhuD (S4) which is involved in both pathways.A total number of15expression vectors have been constructed. Expression of recombinant fusion proteins and enzymatic activity of tagged PhoA were evaluated. Results showed that the expression of recombinant proteins with tagged PhoA and MBP with MrpF were affected by signal peptides S2, S3and S1, respectively. The expression of recombinant proteins of untagged membrane protein MrpF can be enhanced by signal peptides S1and S3. Enzyme activities of whole recombinant proteins indicated that the activities of BL21/pCLE20, BL21/pCLE18and BL21/pCLE10expressing S4-PhoA-MrpF, S3-PhoA-MrpF and S2-PhoA-MrpF increased by multiples. No activities of BL21/pCLE12and BL21/pCLE16expressing PhoA-MrpF and S3-S4-PhoA-MrpF were detected. Enzyme activities of membrane proteins manifest that the activities of BL21/pCLE12, BL21/pCLE16and BL21/pCLE20expressing PhoA-MrpF, S3-S4-PhoA-MrpF and S4-PhoA-MrpF were very low. The relative enzyme activity of pCLE18expressing S3-PhoA-MrpF membrane protein was approximately quadruple that of pCLE10expressing S2-PhoA-MrpF membrane protein. In summary, this work provided useful data of effects of signal peptides on membrane protein localization.