Dataset on recombinant expression of an ancient chitinase gene from different species of Leishmania parasites in bacteria and in Spodoptera frugiperda cells using baculovirus

Abstract

The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.

Keywords: Baculoviruses; Chitinase; Leishmania sp; Recombinant protein; Sf9 insect cell.

Fig. 1

Multiple Leishmania species chitinase amino acid sequence alignment by MUSCLE. a. Signal peptide – box; conserved N-glycosylation signal – underlined; substrate recognition substrate – grey shadow; catalytic domain – black shadow; kinase cAMP dependent domain – black arrow. b. Dendogram of amino acid chitinase sequence distance analysis showing Leishmania species clustering.

Fig. 2

1% agarose gel electrophoresis to analyse the product of L. infantum chagasi chitinase encoding gene expression in amastigotes (A) and promastigotes (P) evaluated by RT/PCR. AC- and PC- correspond to amastigotes and promastigotes negative controls which have RNA treated with Rnase before cDNA synthesis, respectively; M - 1Kbp ladder (Sinapse).

Fig. 3

Expression of L. amazonensis (Am), L. braziliensis (Bz), L. infantum (If) and L. mexicana (Mx) recombinant chitinase produced in the E. coli strain BL21 (D3) at 37°C and 18°C. a. SDS-PAGE stained with PageBlue™, a comassie based gel staining solution purchased from Uniscience; b. Western blotting with anti-histidine antibody detected by colorimetric staining with BCIP/NBT, after hybridization with second antibody labeled with alkaline phosphatase. c. Recombinant protein purified by nickel affinity chromatography from soluble fraction of E. coli strain BL21 (D3), induced for 16hs with 1mM IPTG at 18°C; M and K – Broad range and kaleidoscope pre-stained protein markers from BioRad; Insoluble – pellet of bacteria after lyse; Soluble – soluble fraction of bacteria after lyse; Ni-AChr – nickel affinity chromatography.

Fig. 4

Detergent solubilization assay of L. infantum recombinat chitinase expressed in Spodoptera frugiperda (Sf9) ovarian cells infected with L. infantum chitinase baculovirus. a. Sf9 and Sf9 cells infected with L. infantum chitinase recombinant baculovirus photographed under Neubauer hemocytometer. b. SDS-PAGE stained with PageBlue™; c. Western blotting with the anti-histidine antibody revealed with BCIP/NBT colorimetric reagents for alkaline phosphatase. M: Spectra Multicolor Broad Range Ladder (Thermofisher). IfC – SF9 cells infected with L. infantum chitinase recombinant baculoviruses; X₁₁₄- 1% Triton X114; Igl – 1% Igepal; Chs – 1% CHAPS; T20 – 1% Tween20. Soluble/Insoluble: Soluble and Insoluble fractions of Sf9 cells after detergent and 1% CHAPS treatment.