Prokaryotic expression of DFP1 and DFP2 in Dermatophagoides farinae and their responses to temperature stress

• Published in: Gene Vol. 927; p. 148661
• Main Authors: Wanyu, Zhang, Dongling, Niu, Yae, Zhao, Lianying, Jiao, Chenglin, Guan, Rong, Chai, Li, Hu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.11.2024
• Subjects: Bacterial growth curves; bioinformatics; cold stress; Dermatophagoides farinae; Dermatophagoides farinae proteins; Escherichia coli; genes; heat; Prokaryotic expression; protein content; sequence analysis; stress response; temperature; Temperature stress; Temperature stress; nt; D. farinae; dNTP; kDa; Mw; HSPs; CDS; RNA-seq; OD; kb; E. coli; Bacterial growth curves; Prokaryotic expression; SDS; Amp; CAI; PAGE; Dermatophagoides farinae proteins; ORFs; PMSF; LB; IPTG; DFPs; DFP2; DFP1
• ISSN: 0378-1119; 1879-0038; 1879-0038
• DOI: 10.1016/j.gene.2024.148661

[Display omitted] •DFP1 &DFP2 are highly expressed in D. farinae but their functions are unknown.•DFP1 & DFP2 were expressed in E. coli Rosetta-gami 2 & BL21, respectively.•After temperature stress, recombinant strains grow better than controls.•DFP1 recombinant strains grow better under cold stress than heat stress.•DFP1 & DFP2 have been shown response to temperature stress for the first time. The functions of highly expressed genes DFP1 and DFP2 in Dermatophagoides farinae remain unknown. DFP1 and DFP2 have been abundantly annotated and were up-regulated under temperature stress at 43 °C and –10 °C in our previous RNA-seq study, indicating that DFP1 and DFP2 may have temperature stress response function. Here, we amplified, cloned, and sequenced to obtain the complete coding sequences of DFP1 and DFP2 and predicted their protein characteristics using bioinformatics analysis. Then, prokaryotic expression systems were constructed and found that DFP1 was expressed in Escherichia coli Rosetta-gami 2 (DE3) but not BL21 (DE3); DFP2 was expressed in both BL21 (DE3) and Rosetta-gami 2 (DE3), with higher expression in BL21 (DE3). Finally, the growth curves of bacteria were drawn and indicated that the DFP1- and DFP2-pET32a carrying recombinant bacteria grew better than the respectiveonly pET32a carrying control bacteria after heat and cold stress. This study confirms for the first time that DFP1 and DFP2 respond to temperature stress at the protein level. The constructed prokaryotic expression systems will provide an experimental foundation for future antibody preparation for western blotting detection to confirm the temperature-stress response functions of DFP1 and DFP2.