Expression of CRLAAO in mammalian system

2.4.5.1 Construction of mammalian expression plasmid

The CRLAAO coding region with (S⁺) and without (S^-) native signal sequence was amplified using pTOPOCRLAAO plasmid as template and cloned between BamHI and XbaI sites of the pCDNA3.0HA expression vector in fusion with a N-terminal HA-tag. The transformants were selected and maintained on ampicillin containing medium. The positive clones were analyzed by restriction digestion (Fig. 20). Increased size of the CRLAAOS⁺ fragment was due to the additional 18 amino acids (54 bp) of native signal sequence. The correct orientation

was confirmed by sequencing using the T7 promoter.for primer. HA tag adds an extra streach of 11 amino acids to the N-terminal end of the cloned fragment encoding either CRLAAOS^- or CRLAAOS⁺. Fig. 20 represents the cloning strategy.

Fig. 20. Restriction analysis of mammalian expression vectors. A. Lane 1: 2-log DNA marker (NEB). Lanes 2,3 and 4. pcDNA3.0 vector, pcDNALAAOS^- and pcDNALAAOS⁺ plasmid clones digested with BamHI and XbaI. B. Cartoon depiction of pcDNA expression plasmids showing the PCR fragments cloned between BamHI and XbaI into the mammalian expression vector.

2.4.5.2 Transient transfection of H1299 cells

Transfections were performed using lipofectamine as mentioned in the Methods section. The efficiency of transfection was estimated to be about 30% using a GFP expression vector (Fig. 21). Expression vectors containing CRLAAO cDNA with and without the native sequence were transfected into H1299 cell lines. 24 h after the transfection, culture supernatants and cells were separated by centrifugation.

The soluble and insoluble fractions of the cell lysates were analyzed by Western blotting using αLAAO IgG antibodies. CRLAAO expressed with the native signal sequence was found in the insoluble membrane fraction, however the absence of native signal sequence resulted in the expression of soluble form (Fig. 21 B).

Although the CRLAAO was exported and showed LAAO activity, it could not be easily analysed on western blots as the medium contained high concentrations of serum proteins. HEK cells were also transfected exactly as mentioned above but only the expression construct encoding CRLAAO with the native signal sequence was employed.

Fig. 21. Expression of CRLAAO in H1299 cells. A. Transfection efficiency was estimated to be about 30% using a GFP expression vector. B. Vectors containing CRLAAO ORF with (CRLAAOS⁺) and without native signal sequence (LAAOS^-) were transfected into H1299 cells. 24 h after transfection, soluble and insoluble fractions were separated and analyzed on Western blot using αCRLAAO IgG antibodies. (M): Protein standards (NEB broad range);

(V): Vector; (S^-): Without signal sequence; (S⁺): With signal sequence; (L): CRLAAO positive control.

2.4.5.3 Immuno staining

Immuno staining studies were carried out using α-CRLAAO antibodies inorder to verify the results reported in the previous section. After transfection, H1299 or HEK cells were fixed and the cell membrane was permeabilized as mentioned in the Methods section. The expressed CRLAAO was detected using α-CRLAAO Ab, which was further detected using α-Rabbit Ab conjugated with Cy3 fluorophore (Fig. 22). Nuclei were stained with DAPI. These immuno stained cells were observed under a fluorescence microscope. Merging of images was carried out using Adobe Photoshop. Uniform staining was observed in the cells transfected with the expression construct encoding CRLAAOS^-. However the transfections using pcDNACRLAAOS⁺ resulted in localization of the expressed LAAO at the perinuclear region. In either case no expression was observed in the nucleus.

Fig. 22. Immunostaining of H1299 cells expressing CRLAAO. H1299 cells transiently transfected with pcDNACRLAAO (S⁺/S^-) expression plasmids were stained with αCRLAAO antibodies and counter stained with αRabbit antibodies conjugated with Cy-3 fluorofore.

Nuclei were visualized after staining with DAPI. Mock transfection was performed using the vector alone.

Fig. 23. Expression of CRLAAO in HEK cells. Cells were transfected with mammalian expression plasmid pcDNA CRLAAOS⁺ and expressed LAAO was detected using α-LAAO antibody. Mouse anti-αtubulin antibodies were used for detecting the cell cytoskeleton.

Immunolocalization studies were also carried out in human embryonic kidney (HEK) cell line to verify the above-mentioned observations. HEK cells were transfected with the mammalian expression vector encoding the CRLAAO with its native signal sequence. Cell cytoskeleton was stained using anti α-tubulin antibodies.

Immuno staining of the expressed CRLAAO was performed as mentioned above.

Nearly 30% of the cells were positive for transfection and the expressed CRLAAO seen localizing at the perinuclear region as shown in Fig. 23. Invitro translation was performed with pcDNALAAOS- for further verification of the expression. A strong signal (approx. 55kDa) was observed on the autoradiogram just above p53 (53kDa) which was used as control for the experiment (Fig. 24).

Fig. 24. Invitro translation of CRLAAOS^-. Rabbit reticulolysate translation system was used for the invitro synthesis of the CRLAAO with out its native signal sequence. (See methods section for the details)

2.4.5.4 Activity of Expressed CRLAAO

Culture medium, cellular membrane and soluble fractions were prepared and checked for the LAAO activity using the peroxidase-coupled assay (see Methods section). Transfections were performed using the expression vector alone (Mock), LAAOS^- and LAAO S⁺ constructs. Activity could only be detected in the medium of the cells transfected with the construct encoding CRLAAO with native signal sequence. Only the secreted CRLAAO was shown to be active (Fig. 25). The volumes of the cell lysate fractions were normalized to that of the sample taken from the culture supernatant for the activity assay.

Fig. 25. Activity of CRLAAO expressed in mammalian cells. 24 h after transfection cells were lysed, soluble (Sol) and insoluble membrane pellet (Pel) fractions along with culture supernatant (Sup) were analyzed for CRLAAO activity. Readings from the cell soluble and insoluble fractions were normalized to the volume of culture supernatant.