The production of recombinant human laminin-332 in a Leishmania tarentolae expression system.
Laminin (LM)-332 (alpha3beta3gamma2), a large heterotrimeric glycoprotein, is an essential component of epithelial basement membranes that promotes cell adhesion and migration. Here, we expressed human LM-332 using a novel protein expression system based on the trypanosomatid protozoan host Leishmania tarentolae. Plasmids containing cDNA encoding full-length beta3 and gamma2 subunits and truncated alpha3 subunit were sequentially introduced into L. tarentolae. A recombinant strain harboring the three subunits of human LM-332 efficiently formed heterotrimer and secreted it into the culture medium. Heterotrimeric recombinant LM-332 (rLM-332) could be purified from culture medium with one-step immuno-affinity chromatography. The eluted fraction contained all three subunits, as confirmed by immunoprecipitation and immunoblotting. The purified rLM-332 showed similar cell adhesion activity to rLM-332 purified from mammalian cells, indicating its proper folding and assembly. The obtained expression level was not high; however, we suggest that this expression system has the potential for mass production of LMs for tissue engineering.
Phan
Hoang-Phuong
HP
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Sugino
Marisa
M
Niimi
Tomoaki
T
D000818:Animals; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006801:Humans; D047468:Immunoprecipitation; D007891:Leishmania / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D021122:Protein Subunits; D011994:Recombinant Proteins; D000097794:Kalinin