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. 2024 Jan 9:51:kuae017.
doi: 10.1093/jimb/kuae017.

Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa

Ling Hu  1 , Baicheng Deng  2 , Rong Wu  1 , Miaorong Zhan  1 , Xuchu Hu  2 , Huaiqiu Huang  1
Affiliations

Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa

Ling Hu et al. J Ind Microbiol Biotechnol. .

Abstract

Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB.

One-sentence summary: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.

Keywords: Sporotrix globosa; Cyclophilin B; Optimized expression.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Graphical Abstract
Graphical Abstract
Fig. 1.
Fig. 1.
Prediction of transmembrane region of SgCypB and 3D structure of SgCypB and SgtrCypB. (a) From prediction of transmembrane region, retaining the sequence of outside was the SgtrCypB. (b) In the 3D structure of SgCypB, the dashed area was the SgtrCypB.
Fig. 2.
Fig. 2.
Molecular docking of CsA to SgCypB and SgtrCypB. (a) The 2D representations of SgCypB-CsA interactions. (b) The 3D representations of SgCypB-CsA interactions. (c) The 2D representations of SgtrCypB-CsA interactions. (d) The 3D representations of SgtrCypB-CsA interactions.
Fig. 3.
Fig. 3.
Expression of SgCypB and SgtrCypB protein in different induction time analyzed by SDS-PAGE. (a) Expression of SgCypB protein when induced by 0.5 mM IPTG in 2 hr, 4 hr, and 6 hr in SDS-PAGE. Lane 2h-, 4h-, 6h-: pET-30(a)-SgCypB transformant without IPTG induction at the same time of induction for 2 h, 4 h, and 6 h. Lane 2h+, 4h+, 6h+ : pET-30(a)-SgCypB transformant with IPTG induction for 2 hr, 4 hr, and 6 hr (expression of recombinant protein shown by the arrow). (b) Expression of SgtrCypB when induced by 0.5 mM IPTG in 2 hr, 4 hr, and 6 hr in SDS-PAGE. Lane 2h-, 4h-, 6h-: pET-30(a)-SgtrCypB transformant without IPTG induction at the same time of induction for 2 hr, 4 hr, and 6 hr. Lane 2h+, 4h+, 6h+ : pET-30(a)-SgtrCypB transformant with IPTG induction for 2 hr, 4 hr, and 6 hr (expression of recombinant protein shown by the arrow).
Fig. 4.
Fig. 4.
The colony-forming units of host bacteria when SgCypB and SgtrCypB protein were expressed in different induction time. (a) The CFUs of host cells when pET-30(a)-SgCypB transformant induced with IPTG for 2 hr, 4 hr, 6 hr, and 8 hr showed above the black line of plate, and The CFUs of pET-30(a)-SgCypB transformant without induction at the same time were below the black line. (b) The CFUs of host cells when pET-30(a)-SgtrCypB transformant induced with IPTG for 2 hr, 4 hr, 6 hr, and 8 hr showed above the black line of plate, and The CFUs of pET-30(a)-SgtrCypB transformant without induction at the same time were below the black line. (c) Log 10CFU/mL of pET-30(a)-SgCypB transformant induced with IPTG for 2 hr, 4 hr, 6 hr, and 8 hr. *** refers to significant difference (P < 0.001). (d) OD600 of pET-30(a)-SgCypB transformant induced with IPTG for 2 hr, 4 hr, 6 hr, and 8 hr. (P < 0.001).(e) Log 10CFU/mL of pET-30(a)-SgCypB transformant induced with IPTG for 2 hr, 4 hr, 6 hr, and 8 hr. ns refers to non-significant difference (P > 0.05). (F) OD600 of pET-30(a)-SgCypB transformant induced with IPTG for 2 hr, 4 hr, 6 hr, and 8 hr (P > 0.05).
Fig. 5.
Fig. 5.
Expression and purification of rSgtrCypB protein analyzed by SDS-PAGE and Western blot analysis. (a) Expression and purification of rSgtrCypB protein analyzed by SDS-PAGE Lane M, protein marker; lane 1, pET-30(a) transformant without IPTG induction; lane 2, pET- 30(a) transformant with IPTG induction; lane 3, pET-30(a)-SgtrCypB transformant without IPTG induction; lane 4, pET-30(a)-SgtrCypB transformant with IPTG induction; lane 5, the lysate of recombinant bacteria with IPTG induction; lane 6, the supernatant of the lysate of recombinant bacteria with IPTG induction; lane 7, purified SgtrCypB protein (as pointed by the arrow). (b)Western blot analysis of SgtrCypB protein expression. lane 1, pET-30(a)-SgtrCypB transformant without IPTG induction; lane 2, pET-30(a)-SgtrCypB transformant with IPTG induction; lane 3, purified SgtrCypB protein.
Fig. 6.
Fig. 6.
Peptidyl-prolyl cis-trans isomerase activity test of SgtrCypB with chymotrypsin-coupled assay using double steam water as negative control. * refers to significant difference (P < 0.05), ** refers to significant difference (P < 0.01), *** refers to significant difference (P < 0.001).
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