The Study On Screening Of Peptides That Could Specifically Bind To Sodium Pump α1 Subunit M1-M2 Extramembrane Domain From Phage Display 12 Peptide Library

Sodium pump (Na+, K+-ATPase) is an ion pump that is commonly exists on mammalian cell membrane, which plays an important role in the maintenance of body electrolyte balance, stability of membrane potential and signal transduction. Sodium pump is composed of three different subunits (α,β, andγ) andα-subunit has the catalytic activity and many binding sites of sodium pump regulating factors. Four distinct isoforms (α1,α2,α3 andα4) ofα-subunit have been identified and the basic structure is highly conserved, which is composed of 10 transmembrane domains (M1-M10). The sequences between M1-M2, M1-M2, M5-M6, M7-M8, M9-M10 are located outside the cell membrane, which are called the extramembrane domains and contain a variety of ligand-binding site. Endogenous sodium pump inhibitors, such as ouabain, can interact with the sodium pump by these binding sites, and lead to hypertension through a series of biological effects. Now the first extramembrane domain M1-M2 of sodium pumpα-subunit is identified as an important binding site of sodium pump inhibitor——ouabain. However, in what way ouabain interacts with the sodium pump is uncertain, and whether hypertension can be treated by blocking the binding sites with specific peptide so as to disengage the inhibition of ouabain is still unknown. In this study, the specific binding peptide of sodium pumpα1 subunit was screened from phage display random peptide libraries, which can block or antagonize the inhibition of ouabain to sodium pump. The achievement of the peptide set up a solid foundation for exploring the possible interaction mechanism of sodium pump and its inhibitory factor and the treatment of hypertension.1. Cloning and expression of the sodium pumpα1 subunit M1-M2 proteinObjective: To build sodium pumpα1 subunit M1-M2 extramembrane domain protein expression vector by gene recombination technology, express the protein by IPTG inducing, and use it as the target molecules for the random peptide library screening test.Methods: (1) Construct sodium pumpα1 subunit M1-M2 cloning vector. PCR was carried out with the plasmid YhNα1 (containing human sodium pumpα1 subunit cDNA sequence) as template and two gene fragments of theα1 subunit were obtained: T1T2, T1T2D. The sequence of T1T2 fragment is fromα1 subunit's N terminal to the M2 transmembrane domain and T1T2D fragment is fromα1 subunit's N terminal to M1 transmembrane domain. After tailing, the two gene fragments are connected to pGM-T cloning vector and transformed into E.coli DH5αrespectively. The positive clones were selected and the plasmids pGMT-T1T2 and pGMT-T1T2D were obtained. Sequencing analysis showed that plasmid sequence is consistent with the theoretical sequence. (2) Build the sodium pumpα1 subunit M1-M2 protein expression system. The target gene fragments were cut respectively from T vector, and connected into the expression vector pET32a (+) by double digestion and transformed into E. coli Rosetta2. The positive clones were selected and the expression plasmids pET32a (+)-T1T2 and pET32a (+)-T1T2D were obtained. After verifying the sequence of the target gene by double digestion, the expression bacteria were induced by IPTG with a final concentration of 0.4mM/L and SDS-PAGE was carried out to detect the expression of fusion protein.Results: (1) PCR was carried out with the plasmid YhNα1 as template and two gene fragments of theα1 subunit were obtained successfully: T1T2, T1T2D. 1% agarose gel electrophoresis showed that the PCR products were in the right positions, which indicated that the amplification of gene fragments were successful. After tailing, the PCR productions are connected to pGM-T vector and transformed into E.coli DH5α. The positive clones were amplified and plasmids were extracted. Restriction enzyme digestion and sequencing results showed that plasmid sequences are consistent with the theoretical sequences. (2) The two gene fragments were linked to pET32a (+) expression vector which was double-digested with the same enzyme and transformed into Rosetta2 competent cells respectively. Recombinants were screened on the plate containing Amp. Double-digestion results showed that the protein expression systems were constructed successfully. (3)T1T2 and T1T2D fusion protein were expressed respectively in Rosetta2 by IPTG induction. SDS-PAGE results showed that T1T2D fusion protein was expressed successfully while the T1T2 fusion protein was not expressed.Conclusion: The cloning vectors and expression vectors of T1T2 and T1T2D were constructed with genetic engineering technology. T1T2D fusion protein was expressed successfully while T1T2 fusion protein was not expressed. The reason may be that T1T2 fragment contains transmembrane sequence and has high hydrophobicity.2. Screening the binding peptide of the sodium pumpα1 subunit M1-M2 extramembrane domainObjective: Screen the specific binding peptides from phage display random peptide libraries with M1-M2 extramembrane domain peptide as a target, which can block or antagonize the inhibition of ouabain to sodium pump. And the obtaining of the peptide would lay a solid foundation for the further exploring the possible interaction mechanism of sodium pump with its inhibitory factor and the treatment of hypertension.Methods: (1) Screen specific binding peptides from the phage random 12-peptide library with solid-phase panning method. Three turns "adsorption - elution - adsorption" panning were performed with the synthetic sodium pumpα1 subunit M1-M2 peptide (ATEEEPQNDNLGGGS) as the target molecules, and the phage display peptides that can bind with the target protein were enriched. (2) double-sandwich ELISA identify positive phage clones: The sodium pumpα1 subunit M1-M2 extramembrane domain peptide (ATEE EPQNDNLGGGS)is used to coated ELISA plate and the negative control was set at the same time. Add the amplified phage clone to the board, and then add HRP - Anti-M-13 antibody and measure OD values at 420nm. (3) The concentration-dependent experiments: The sodium pumpα1 subunit M1-M2 extramembrane domain peptide (ATEEEPQNDNLGGGS) was coated as the target protein. Every clone prepares a row of holes to be coated. Phage clones are amplified and phage titer was determined. About 1012 pfu phages were added to plate and serially diluted phages by five-fold were added, too. Then HRP - Anti-M-13 antibody were added and OD values were measured at 420nm. The relationship between the concentration and combining power of phage clone was observed by plotting the value of absorbance (Y-axis) against the dilution multiple (X-axis). (4) The extraction and sequence analysis of M-13 phage ssDNA: The blue positive colonies were selected randomly from the third turns screening plate, and the phages were amplified in E.coli ER2738 host bacteria. The supernatant was precipitated by PEG/NaCl solution and the phage ssDNA was extracted by NaI/ethanol solution. After sequencing, the corresponding amino acid sequence was deduced according to "genetic code table" in phage display 12 peptide library kit instructions. (5) Ouabain competitive inhibition test: 96-well plates were coated by sodium pumpα1 subunit M1-M2 extramembrane domain peptide (ATEEEPQNDNLGGGS). Ouabain solution with the concentration of 80μg/ml, 40μg/ml, 20μg/ml, 10μg/ml, 5μg/ml, 2.5μg/ml. was mixed respectively with the phage supernatant by 1:1. Then the mixed liquors were added into 96-well plates and OD values were measured. And the rate of inhibition was calculated.Results: (1) Phage display 12 peptide library screening results: Through the output/input ratio of each round of screening, it is observed that the recovery rate of phage was gradually increased with obvious enrichment effects. (2) Double-sandwich ELISA test: Nine phage mono-clones were identified from the random phage clones by comparing OD values of the experimental hole to the negative hole. (3) The concentration-dependent test: The nine phage clones screened from double-sandwich ELISA test were tested by the concentration-dependent experiments. The results showed that only four phage clones meet the requirements. (4) The extraction and sequence analysis of M-13 phage ssDNA:. The results showed that the molecular weight of extracted ssDNA is consistent with M-13 phage DNA. Sequencing analysis showed that the amino acid sequences of the four phage clones display peptides are identical, which are: WHWRNPDFWYLK. (5) Ouabain competitive inhibition test: The results showed that the inhibition rate to phage display peptide was decreasing with the reduction of ouabain, which indicated that the display peptide can bind specifically with M1-M2 extramembrane domain and can block or antagonize the inhibition of ouabain to sodium pump.Conclusion: A specific binding peptide (WHWRNPDFWYLK) was successfully obtained from phage-displayed random peptide library with synthetic M1-M2 extramembrane domain peptide as a target, which can inhibit competitively the binding of ouabain to sodium pump. The obtaining of the binding peptide laid a solid foundation for the further exploring the possible interaction mechanism of sodium pump with its inhibitory factor and the treatment of hypertension.