Sequence analyses of CpLTL. (A) Domain organization of CpLTL protein using SMART. SP: signal peptide, TM: transmembrane domain. (B) Amino acid sequences of apicomplexan parasite LTLs were aligned with CpLTL using the Clustal W algorithm. The metal-binding sites were marked with a red arrow, and the carbohydrate-binding sites were marked with a black arrow. Amino acid identities with orthologs from other Cryptosporidium species and apicomplexan parasites.

Expression of recombinant CpLTL in Escherichia coli and identification of native CpLTL in C. parvum sporozoites. (A) PCR amplification of the CpLTL of C. parvum. Lane M: 2000 bp DNA ladder maker. Lane 1: CpLTL product. (B) Purity of recombinant CpLTL assessed by SDS-PAGE analysis. Lane M: molecular weight markers, Lane 1: CpLTL purified using Glutathione Sepharose affinity chromatography. (C) Expression of recombinant CpADF in E. coli determined via western blotting analysis using an anti-GST tag. Lane M: protein size makers. Lane 1: recombinant CpADF protein. (D) Western blotting analysis of native CpLTL in C. parvum sporozoites using pre-immune serum (left panel) and polyclonal antibodies (right panel). Lane M: protein size makers. Lane 1 and lane 2: native protein from sporozoites.

Identification of host proteins interacting with CpLTL by GST-pull down combined with LC-MS/MS. (A) Silver-stained one-dimensional SDS-PAGE analysis of GST-CpLTL. Line M: marker. Line 1: GST-CpLTL. Line 2: GST control. Line 3: PBS control. (B) The Venn diagram of the differentiated proteins was obtained by LC-MS/MS identification.