Inhibition of protein expression by the interaction of G-Quadruplex and RHAU peptide in E. coli

Science & Technology Development Journal, 22(4):378-384 Open Access Full Text Article Original Research 1Center for Bioscience and Biotechnology, University of Science, National University-HCMC, Vietnam 2Institue of Tropical Biology, VAST 3Faculty of Biotechnology, Ho Chi Minh City Open University, Vietnam Correspondence Dung T Dang, Center for Bioscience and Biotechnology, University of Science, National University-HCMC, Vietnam Faculty of Biotechnology, Ho Chi Minh City Open University, Vietnam Email: dung.dthanh@ou.edu.vn History  Received: 2019-10-01  Accepted: 2019-12-24  Published: 2019-12-31 DOI : 10.32508/stdj.v22i4.1712 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Inhibition of protein expression by the interaction of G-Quadruplex and RHAU peptide in E. coli Tuom T.T Truong1, ThuM.T. Dao1, Trang P.T Phan1, Hoang D Nguyen1, Dung H Nguyen2, Dung T Dang1,3,* Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: G-quadurplex (G4) formation plays a role inmany biological processes such as repli- cation, transcription, translation, and telomeric maintenance. Stabilization of G4 structure by pep- tide has recently emerged as a potential approach in the regulation of protein expression. In this study, we reported on regulation of cyan fluorescent protein (CFP) expression by the interaction of G4 and RNA helicase associated with AU-rich elements (RHAU) peptide in E. coli. Methods: A se- quence of TTGGGTGGGTGGGTGGGT (formed into G4 structure) was genetically applied to cfpgene as a reporter gene (g4-cfp). Both g4-cfp andDHX36 (or DDHX36) genes were cloned to pET-Duet1 vector that allowed to simultaneously express both G4-CFP protein and RHAU (or DRHAU) protein under IPTG inducer. Results: The level of G4-CFP expression in the presence of RHAU (pD64) was around 2-fold and 3-fold lower than that of G4-CFP expression in the presence of DRHAU (pD65) and G4-CFP expression alone (pD21), respectively. Conclusion: RHAU might selectively bind G4 structure of mRNA of G4-CFP, resulting in inhibition of G4-CFP expression in E. coli. The G4 and RHAU peptide interaction would provide a promising approach for inhibition of gene expression in many biomedical applications. Key words: G4-RHAU interaction, Inhibition, Protein expression INTRODUCTION G-quadruplexes (G4s) are G-rich sequences that can fold into four single-strandedDNAorRNA structures by hoogsteen hydrogen interaction1 (Figure 1). G4 structures can be parallel or nonparallel topology 2. In the cell, the formation of G4 structure plays a cru- cial role in many biological processes such as replica- tion, transcription, translation and telomeric mainte- nance3. For instance, the formation of G4 in the un- translated region (UTR) that significantly affected the level expression of protein4–6. Formation ofG4 struc- ture at the telomeric region that can prevent binding of telomerase to its DNA target leads to inhibition of telomeric elongation7–9. Therefore, the formation of G4s in DNA or RNA is considered to be a newmolec- ular target for molecules in cancer therapeutics 10–12. Specific recognition and stabilization of G4 by pep- tides have emerged as a potential approach for reg- ulation of many biological processes due to the fol- lowing advantages such as (i) peptides are easier to design and synthesize than recombinant proteins, (ii) peptides canmimic the interaction of G4 and protein. Recently, specific recognition of parallel G4 by RHAU has been reported13. The full length of RHAUprotein consisting of 1008 aa can bind parallel G4 and unwind G4 structure in the presence of ATP in the cells. How- ever, only N-region of RHAU peptide (without heli- case domain) can specifically bind and stabilize par- allel G4 structure13. Insight into the structure of G4 and RHAUpeptide showed the RHAU covers a termi- nal guanine base and binds the parallel G4 using 3 an- chor point electrostatic interactions between negative charge of phosphate groups and 3 positive charges of amino acids14. The studies of the interaction of G4 and RHAU peptide for biological applications have also been reported. Incorporating RHAU peptide to CFP allowed to generate the fluorescent probe which can visualize and distinguish G4 topologies 15. De- velopment of the novel ribonuclease by fusing RHAU with RNase H catalytic domain that can selectively bind G4 and cleave RNA in RNA:DNA hybrid at the specific manner16. However, the application of stabi- lization of G4 by RHAU peptide in inhibition of pro- tein expression has not been reported yet. Herein, we study inhibition of CFP expression by the interaction between G4 and RHAU peptide (140 aa) in E. coli (Figure 2). Two genes: g4-cfp (consisting G4 structure) and DHX36 were genetically cloned in the pET-Duet1 vector that allows expressing G4-CFP and RHAU simultaneously. The presence of RHAU Cite this article : T.T Truong T, M.T. Dao T, P.T Phan T, D Nguyen H, H Nguyen D, T Dang D. Inhibition of protein expression by the interaction of G-Quadruplex and RHAU peptide in E. coli. Sci. Tech. Dev. J.; 22(4):378-384. 378 Science & Technology Development Journal, 22(4):378-384 Figure 1: A) G4 structure is formed in the presence of cation K+ or Na+. B) G4s with different topologies: parallel and non-parallel2. peptide might stabilize G4 structure of mRNA of G4- CFP, resulting in inhibition of the amount of G4-CFP expression inE. coli. Thatwould open a potential pep- tide candidate for many biomedical applications. MATERIALS - METHODS Construction of plasmids G4 sequence was applied to the upstream of cfp gene (Figure 3). DNA encoding for this CFP protein was amplified by PCR using pHT58217,18 containing cfp gene as the template with a pair of primer: forward primer ON1: 5’-gcgtagatctgttgggtgggtgggtgggtatgg gcgtgagcaagggcgaggagctgttc-3’ and reverse primer ON2: 5’-ccatctcgagttacttgtacagctcgtccatgccgagagtg-3’ (IDT, Singapore). This PCR product was then cloned into treated pET-Duet1 (containing two T7 promot- ers) at BglII/XhoI (New England Biolabs, United Kingdom) sites, resulting in plasmid pD21 (consist- ing of G4 structure next cfp gene). Plasmids pD64 (expressing both G4-CFP and RHAU) and pD65 (expressing both G4-CFP and DRHAU-without RSM motif) were generated by cloning DHX36 and DDHX36 to the second multi- cloning site of pD21, respectively. DNAs encoding for RHAU and DRHAU were amplified by PCR using RHAU as the template with ON3/ON5 (ON3: 5’-gcgtggatccgtccatgcatcccgggcacctgaaag-3’, ON5: 5’-gtgtaagcttctagccgctttttttcttttg-3’) and ON4/ON5 (ON4: 5’-gtgtggatccgaaacaggggcagaagaacaag-3’) (IDT, Singapore), respectively. PCR products (DHX36 and DDHX36) were then cloned into treated pD21 at Bam HI/Hind III (New England Biolabs, UK) sites, resulting in pD64 and pD65, respectively. Co-expression of protein in E. coli The plasmid pET-Duet1 containing two T7 promot- ers that allow expressing two proteins simultaneously. Plasmids pD21 (g4-cfp), pD64 (g4-cfp and DHX36) and pD65 (g4-cfp and DDHX36) were transformed into the host of E. coli strain Rossetta (DE3) plysS. The bacteria were cultured in LB medium contain- ing ampicillin at 37oC, 200 rpm. When reaching an OD600 of 0.6, IPTG (Sigma Aldrich, Singapore) was 379 Science & Technology Development Journal, 22(4):378-384 Figure 2: Schematic representation of inhibition of protein expression by RHAU and G4 interaction in E. coli. RHAU peptide and G4-CFP are separately expressed in E. coli, RHAU peptide selectively binds and stabilizes G4 structure of mRNA of G4-CFP, resulting in inhibition of G4-CFP expression. Figure 3: mRNA sequence of g4-cfp. Start codon (aug) and stop codon (uaa) are in bold, G-rich sequence (ggguggguggguggg), which formed into RNA G4 structure is in bold and underline, andmRNA sequence of cfp is in italic. added to a final concentration of 0.3 mM. Then the cells were incubated overnight at 16oC, 250 rpm be- fore being harvested. Evaluation of CFP expression by spec- trophotometry and statistical analysis The cells were harvested at OD600 of 1.2. The pellets were re-suspended into the bugBuster protein extrac- tion reagent (Merck, Singapore) plus bezonase nu- clease to degrade DNA and RNA. The insoluble de- bris was removed by centrifugation at 20,000 rpm, 4oC.The soluble fraction of proteins was evaluated by the spectrophotometry at the excitationwavelength of 410 nm and emission wavelength of 475 nm. Statisti- cal analysis for the determination of p-value between protein expression levels was calculated in excel. Evaluation of protein expression by SDS- PAGE and statistical analysis The pellets of cells were re-suspended into lysis buffer (Tris-HCL 20 mM, sucrose 15%, pH7.4). The solu- ble fractions were added into loading dye buffer (2% SDS, 100 mM DTT, 10% glycerol, 50 mM Tris-HCL and 0.1% bromophenol blue dye pH 6.8). The sam- ples were then heated at 95ºC in 5 minutes. The insol- uble debris was removed by centrifugation at 13,000 rpm in 5 minutes. The soluble fraction of protein was applied to the denaturing polyacryamid gels for electrophoresis. The gels were then visualized with coomassie brilliant blue. The level of protein expres- sion was analyzed by the AlphaEaseFC software. Sta- tistical analysis for the determination of p-value be- tween protein expression levels was calculated in ex- cel. RESULTS 380 Science & Technology Development Journal, 22(4):378-384 Construction of plasmid, protein expres- sion DNA sequence of genes in plasmids pD21, pD64 and pD65 have been confirmed by DNA sequencing (1st BASE, Singapore). The plasmids pD64 or pD65 con- taining twoT7 promoters allowed expressing simulta- neously G4-CFP and RHAU or G4-CFP and DRHAU under IPTG inducer (Figure 4). All the proteins were expressed in E. coli under IPTG regulation. Evaluation of CFP expression by spec- trophotometry The level of CFP protein expression in E. coliwas eval- uated by spectrophotometry. The plasmid pD21 only bearing g4-cfpwas expressed in E. coli under IPTG in- ducer. The fluorescent intensity of pD21was observed around 490 a.u and the fluorescent intensity of pD64 and pD65 were observed around 156 a.u and 386 a.u, respectively (Figure 5). Statistical analysis also showed all p-values were <0.05 that determined there was not a significant difference between the means of two group samples of protein expression. The yield of G4-CFP expression of pD65 was 1.2 times lower than that of pD21. It would explain that two pro- teins (G4-CFP and DRHAU) were expressed simul- taneously; therefore, DRHAU affects the yield of G4- CFP expression of pD65. Interestingly, the yield of G4-CFP expression of pD64 in the presence of RHAU was around 2-fold and 3-fold lower than that of G4- CFP expression of pD65 and pD21, respectively. The presence of RHAU peptide might stabilize G4 struc- ture of mRNA of G4-CFP, resulting in inhibition of yield of G4-CFP expression in E. coli. In contrast, DRHAU (without G4 binding domain) does not bind G4 in mRNA of G4-CFP, therefore DRHAU was the minor effect of the yield of G4-CFP expression. Evaluation of protein expression by SDS- PAGE The expression of G4-CFP, RHAU and DRHAU were visualized by SDS-PAGE (Figure 6). The result showed both G4-CFP and RHAU of pD64 and both G4-CFP and DRHAU of pD65 were observed on the SDS-PAGE that clarified two T7 promoters of pET- Duet1 can control separately the expression of two proteins in E. coli under IPTG inducer. Data also showed a difference in expression level of RHAU (pD64) and DRHAU (pD65) was negligible. There- fore, RHAU specific motif (RSM) of RHAU was not affected by the expression of RHAU. Interestingly, the analysis of intensity by the AlphaEaseFC software showed the level of G4-CFP expression of pD64 was 2 times lower compared to the level of G4-CFP expres- sion of pD65. That is in line with the evaluation of G4-CFP expression of pD64 and pD65 by spectropho- tometry. The expression of RHAU peptide of pD64 might selectively bind and stabilize theG4 structure of mRNAofG4-CFP, resulting in inhibition of the trans- lational process of ribosomes to mRNA of G4-CFP. DISCUSSION Formation of G4 plays a role in many biological pro- cesses such as replication, transcription, translation and telomeric maintenance. The stabilization of G4 structure by peptide has recently emerged as a poten- tial approach in the regulation of protein expression. RHAU peptide can selectively bind and stabilize G4 structure via electrostatic interactions between nega- tive charge of phosphate groups and 3 positive charges of amino acids. That allows RHAUpeptide to be a po- tential candidate for the study of stabilization of G4 in many biological processes. Although RSM (16 aa) can be sufficient for specific binding to G4, a lower bind- ing affinity at micromolar range that prevents to study of G4 and RSM interaction in biology. The length of the peptide significantly influences the affinity of binding. The RHAU peptide with 140 aa (consisting of RSM) which can selectively bind G4 at nanomo- lar range15. The RHAU peptide can selectively bind and stabilize both DNA and RNA G4s that have been studied in the development of the fluorescent probes specific for G4 topologies and ribonuclease for pro- grammable RNA cleavage. Herein, the RHAU (140 aa) selectively binds RNA G4 of mRNA that may pre- vent the ribosomes from sliding over mRNA. Using vector pET-Duet1 containing two T7 promoters at different regions allows this vector to control the over- expression of two proteins separately. Expression of a single protein being cloned into this vector often yields higher level than co-expression. That explains the expression of a single G4-CFP protein is observed at slightly higher level than co-expression of G4-CFP and DRHAU. Interestingly, the level of G4-CFP ex- pression was significantly decreased in the presence of RHAU compared to the level of a single G4-CFP expression and G4-CFP expression in the presence of DRHAU. That explains the presence of RHAU may inhibit protein expression of G4-CFP by the interac- tion of G4 (mRNA of CFP) and RHAU. In contrast, DRHAU without RSM can not recognize and bind to G4 structure that does not affect the translational pro- cess of ribosomes toG4-CFP.The inhibition of protein expression by G4-stabilized RHAU will be well char- acterized in the vector containing two different pro- moters. 381 Science & Technology Development Journal, 22(4):378-384 Figure 4: Construction of plasmids pD21 (containing G4-CFP alone), pD64 (containing G4-CFP and RHAU) and pD65 (containing G4-CFP and DRHAU). Figure 5: Evaluation of CFP expression by spectrophotometry. G4-CFP expression of pD21 was observed around 490 6.5% a.u, G4-CFP expression of pD64 and pD65 were observed at 156 10% a.u and 386 8.2% a.u, respectively. P-value between pD21 and pD64 is 0.0024, p-value between pD21 and pD65 is 0.018. All experiments were performed in the triplicate. CONCLUSIONS Stabilization of G4 structure by RHAU peptide inhib- ited expression of G4-CFP in E. coli. G4-CFP and RHAU or G4-CFP and DRHAU were able to sepa- rately express in E. coli under IPTG inducer. The yield of G4-CFP expression of pD64 in the presence of RHAU was around 2-fold and 3-fold lower than that of G4-CFP expression of pD65 and pD21, respec- tively. RHAU peptide might selectively bind and sta- bilize G4 structure of mRNA of G4-CFP, resulting in inhibition of G4-CFP expression. The G4 and RHAU peptide interaction would provide a promising ap- proach for inhibition of unexpectable protein expres- sion in cells. ABBREVIATIONS CFP: Cyan Fluorescent Protein G4: G-quadruplex 382 Science & Technology Development Journal, 22(4):378-384 Figure 6: A) Analysis of protein expression by SDS-PAGE. The samples of protein ladder (M), pD64 before (-), and after inducing IPTG (+), pD65 before (-) and after inducing IPTG (+) were applied to the denatur- ing polyacrylamid gel for electrophoresis. CFP (28 kDa), RHAU (18.5 kDa) and DRHAU (16.5 kDa) were all visualized in the gel by coomassive briliant blue solution. B) The relative integrated intensity value of pro- teins (after addition of IPTG) were analyzed by the AlphaEaseFC programe: integrated density value (IDV) of pD64 (G-CFP around 3557 10%IDV, RHAU around 3392 8.8% IDV), pD65 (G-CFP around 71346.7% IDV,DRHAUaround304111% IDV). p-valuebetweenG4-CFPandRHAUofpD64 is 0.037,p-valuebetween G4-CFP and DRHAU of pD65 is 0.004. All experiments were performed in the triplicate. 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