Recombinant protein preparation and functional analysis of mannan-binding protein

The human MBP cDNA

A vaccinia virus transfer vector, pBSF-2-16

Wild-type vaccinia virus strain Western Reserve (WR) and its isatin-β-thiosemicarbazone-(IBT)-dependent derivative

RK13 (a rabbit kidney cell line, ATCC CCL 37), COS-7 (an African green monkey kidney cell line, ATCC CRL 1651), and HLF (a human hepatoma cell line, JCRB 0405)

All materials for cell culture, virus proliferation, vector construction and transfection, virus purification, and recombinant protein purification

An affinity column of mannan-Sepharose 4B and gel filtration chromatography with Sephacryl S-300

Loading buffer: 20 mM of imidazole, 1.25 M NaCl, and 20 mM of CaCl₂, pH 7.8

Elution buffer: 20 mM of imidazole, 1.25 M NaCl, and 4 mM of EDTA, pH 7.8

All chemicals for gel electrophoresis

All reagents for MBP complement activation assay

Gelatin-veronal buffer (GVB): 5 mM of veronal buffer, pH 7.4, containing 0.145 M NaCl, 0.1% gelatin, 2 mM of CaCl₂, and 0.5 mM of MgCl₂

Additional reagents and equipment for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), immunodetection, affinity column purification, gel filtration chromatography, and MBP complement activation assay

Construction of a vaccinia virus transfer vector and recombinant vaccinia virus (RVV) (Note 1)

a.

The human MBP cDNA is excised from the vector by digestion with SmaI and SacI and is subcloned into a vaccinia virus transfer vector, pBSF2-16.

b.

In the resultant transfer vector (pBSF2-16/MBP), the human MBP cDNA is located immediately downstream of the A-type inclusion body of a cowpox virus (ATI) hybrid promoter, and the human MBP cDNA flanked by the ATI hybrid promoter is interposed in the hemagglutinin (HA) gene, a selection marker for obtaining RVVs.

c.

pBSF2-16/MBP (5 µg) and intact genomic DNA (10 µg) extracted from wild-type WR vaccinia virus are diluted in 1.5 mL of OptiMEM, and 30 μg of a cationic liposomal transfection reagent is diluted in 1.5 mL of OptiMEM. These two solutions are then mixed gently and incubated at room temperature for 45 min to yield the DNA-liposome complex.

d.

Cultured COS-7 cells (2.5 × 10⁵ cells in a 10-cm dish) are infected with 2.5 × 10⁴ plaque-forming units of IBT-dependent vaccinia virus for 1 h. The cells are exposed to the transfection solution for 8 h at 37°C under 5% CO₂.

e.

After 20 h of incubation, the cells are harvested, and then the virus progeny is released by sonication. Monolayer culture of RK13 cells is infected with the progeny virus.

f.

The virus progeny obtained from HA-negative plaques inoculated onto monolayer culture of RK13 cells and further screened for the expression of human MBP by immunohistochemical staining with a monoclonal antibody specific for human MBP.

g.

The virus progeny obtained from MBP-positive plaques is selected once more to purify the RVV.

Expression and purification of recombinant human MBP

a.

HLF cells at a density of 1 × 10⁷ cells per 75 cm² are infected with the RVV containing the human MBP cDNA at a multiplicity of infection of 5, and then the infected cells are incubated for 48 h at 37°C under 5% CO₂ (Note 2).

b.

The culture supernatant of HLF cells is harvested and centrifuged to remove cell debris and viral particles.

c.

The resulting supernatant is mixed with a half volume 3 × loading buffer, filtered through a 0.45-μm filter, and then applied to a mannan-Sepharose 4B column equilibrated with loading buffer.

d.

After washing the column with loading buffer, the bound recombinant proteins are eluted with elution buffer.

e.

The eluted fractions are pooled and concentrated. The purity of the recombinant MBP can be checked by SDS-PAGE under reducing conditions, and the degree of oligomerization is assessed by SDS-PAGE on a 3%–10% gradient gel under nonreducing conditions. A typical yield of recombinant human MBP is 5 mg/L of culture medium.

Assay for complement activation activity of MBP by passive hemolysis

a.

The sheep erythrocyte suspension is centrifuged at 350 ×g for 5 min at 4°C and suspend with saline several times.

b.

A portion of the suspension is taken, and the erythrocyte is lysed with water.

c.

Determine the cell density of an erythrocyte suspension in (a) by measuring the absorbance at 541 nm.

d.

Mix 1 mL of 200 μg/mL mannan solution with 1 mL of a 0.5 mg/mL CrCl3 solution.

e.

The mixture is added to 2 mL of a 1 × 10⁹ cells/mL erythrocyte suspension, followed by incubation for 5 min at room temperature with occasional mixing.

f.

The reaction is stopped by adding 3 mL of ice-cold GVB buffer.

g.

The resulting erythrocytes coated with mannan (mannan-erythrocytes, ME) are washed with the GVB buffer thrice and then resuspended with GVB buffer at a density of 1 × 10⁹ cells/mL.

h.

Mix 0.1 mL of ME suspension and 0.4 mL of recombinant human MBP diluted with GVB buffer and incubated with gentle shaking to sensitize ME with MBP.

i.

The ME suspension is then washed with ice-cold GVB buffer and resuspended at a density of 1 × 10⁹ cells/mL. Two volumes of CH50 of MBP-depleted guinea pig complement (Note 3), 0.1 mL of the ME suspension sensitized with MBP, and GVB buffer are mixed in a total volume of 1.5 mL on ice and are then incubated for 1 h at 37°C.

j.

After the reaction mixture has been centrifuged, the absorbance at 541 nm of the supernatant is measured. Maximal lysis is obtained by incubation of 0.1 mL of the ME suspension with 0.14 mL of water. The degree of specific lysis is expressed as a percentage of the maximal lysis (Note 4).

Vaccinia viruses are DNA viruses that infect almost all mammalian cells. The use of vaccinia virus as a vector to introduce cDNAs into mammalian cells has several useful advantages, including a relatively high level of protein synthesis, proper folding, disulfide bond formation, glycosylation, and other post-translational modifications. Thus, a vaccinia virus expression system is useful for the recombinant preparation of highly assembled macromolecules, such as MBP.

Recombinant human MBPs have been produced in myeloma cells, COS cells, and CHO cells. However, these recombinant MBPs contain oligomers with fewer polypeptide chains than those found in native MBP and exhibit less ability to active complement compared to native MBP.

A CH50 unit is defined as the amount of complement solution that gives a 50% hemolysis of 1 × 10⁹ cells/mL erythrocytes.

The complement activation activity of MBP is assayed by passive hemolysis using mannan-coated sheep erythrocytes in the presence of complement. The binding of MBP to the surfaces of mannan-coated erythrocytes triggers the complement cascade, resulting in the formation of membrane attack complexes and cell lysis. The degree of lysis is determined by hemoglobin colorimetry. The complement activation activity of recombinant human MBP increases with the degree of oligomerization.