Prokaryotic Expression and Bioinformatic Analysis of Rv3432c From Mycobacterium tuberculosis

YI Haibo, GAO Xinghong, LUO Guo, XU Peng, WANG Huan

Abstract

Objective To express the protein enconded by the Rv3432c gene of Mycobacterium tuberculosis (M.tb) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M.tb.
Methods The Rv3432c gene was amplified by PCR using the genomic DNA of the inactivated M.tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL.
Results pET-28a-Rv3432c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×103, which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of －0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured.
Conclusion We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M.tb.

 

Keywords: Mycobacterium tuberculosis, Rv3432c protein, Prokaryotic expression, Bioinformatics

 

Full Text:

PDF


References


WHO. Global tuberculosis report 2023. (2023-11-07)[2024-02-09]. https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-report-2023.

STRAUSBAUCH P H， FISCHER E H. Chemical and physical properties of Escherichia coli glutamate decarboxylase. Biochemistry，1970，9(2): 226–232. doi: 10.1021/bi00804a006.

SHIN S M， KIM H， JOO Y， et al. Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity. J Agric Food Chem，2014，62(50): 12186–12193. doi: 10.1021/jf504656h.

HUANG J， FANG H， GAI Z C， et al. Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: crystal structure and functional analysis. Biochem Biophys Res Commun，2018，503(3): 1703–1709. doi: 10.1016/j. bbrc.2018.07.102.

SAGONG H Y， SON H F， KIM S， et al. Crystal structure and pyridgoxal 5-phosphate binding property of lysine decarboxylase from Selenomonas ruminantium. PLoS One，2016，11(11): e0166667. doi: 10.1371/journal. pone.0166667.

MARAS B， SWEENEY G， BARRA D， et al. The amino acid sequence of glutamate decarboxylase from Escherichia coli. Eur J Biochem，1992，204: 93–98. doi: 10.1111/j.1432-1033.1992.tb16609.x.

LIU T L， WANG L， ZHOU Y， et al. Prokaryotic expression and protein bioinformatic analysis of the Mycobacterium tuberculosis Rv1787 gene encoding the protein PPE25. J Pathog Biol，2019，14(7): 765–772. doi: CNKI:SUN:ZISC.0.2019-07-006.

CHEN Z Z， KAN J Q， SHI H. Recent progress in molecular mechanism of acid resistance in Escherichia coli. Food Science，2015，36(21): 273–278. doi: 10.7506/spkx1002-6630-201521051.

PENG C L， HUANG J， ZHAO W R， et al. Effects of acid stress on the expression of key genes of glutamate decarboxylase system and enzyme activity in Lactobacillus brevis. J Chem Eng Chin Univ，2015，29(2): 359–365. doi: 10.3969/j.issn.1003-9015.2015.02.017.

FREDDI L， DAMIANO M A， CHALOIN L， et al. The glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella. Front Microbiol，2017，8: 2236. doi: 10.3389/fmicb.2017.02236.

BOONBURAPONG B， LALOKNAM S， INCHAROENSAKDI A. Accumulation of gamma-aminobutyric acid in the halotolerant cyanobacterium Aphanothece halophytica, under salt and acid stress. J Appl Phycol，2016，28(1): 1–8. doi: 10.1007/s10811-015-0523-7.

TRAMONTI A， VISCA P， De CANIO M， et al. Functional characterization and regulation of gadX, a gene encoding an AraC/XylS-like transcriptional activator of the Escherichia coli glutamic acid decarboxylase system. J Bacteriol，2002，184(10): 2601–2613. doi: 10.1128/JB.184.10.2603-2613.2002.

BRAUN H S， SPONDER G， ASCHENBACH J R， et al. The GadX, regulon affects virulence gene expression and adhesion of porcine enteropathogenic Escherichia coli in vitro. Vet Anim Sci，2017，3: 10–17. doi: 10.1016/j.vas.2017.04.001.

RUPAL R， VINAYAK S， BIJINA J M， et al. Mycobacterial response to an acidic environment: protective mechanisms. Pathog Dis，2022，80(1): ftac032. doi: 10.1093/femspd/ftac032.

NGUYEN L， PIETERS J. The trojan horse: survival tactics of pathogenic mycobacteria in macrophages. Trends Cell Biol，2005，15(5): 269–276. doi: 10.1016/j.tcb.2005.03.009. kinase g in growth and glutamine metabolism of Mycobacterium bovis BCG. J Bacteriol，2005，187(16): 5852–5856. doi: 10.1128/JB.187.16.5852-5856.2005.

LEE J J， LIM J， GAO S， et al. Glutamate mediated metabolic neutralization mitigates propionate toxicity in intracellular Mycobacterium tuberculosis. Sci Rep，2018，8: 8506. doi: 10.1038/s41598-018-26950-z.

YAO S， LIANG P， TU Q X， et al. Bioinformatics-based study of key genes for ferroptosis in the mechanism of acute myocardial infarction. J Zunyi Med Univ，2023，46(4): 357–364. doi: 10.14169/j.cnki.zunyixuebao. 2023.0053.

ZUO Q Q， QIAN L， WEN W E， et al. Bioinformatics analysis of the structure and function with catalase protein from Senecio scandens Buch. -Ham. ex D. Don. J Zunyi Med Univ，2021，44(1): 42–47. doi: 10.14169/j. cnki.zunyixuebao.2021.0008.


Refbacks

  • There are currently no refbacks.