Expression of human mannan-binding lectin in Pichia pastoris (B171)

Mannan-binding lectin (MBL), a pattern-recognition molecule binds to sugars present on the cell surface of microorganisms and activates the lectin pathway of complement. Three naturally occurring point mutations that occur in exon 1 of MBL 2 gene give rise to the structural variants of MBL and are correlated with impaired immune response. Wild type human MBL A and its variant, MBL B (Gly to Asp) were expressed in the yeast, Pichia pastoris. The yeast system has several advantages such as easy to manipulate, less expensive and generally gives high expression levels. Western blot analysis under reduced conditions of rMBL A and rMBL B expressed in the yeast culture medium exhibited a molecular size similar to that of native MBL (32 kDa) purified from human plasma (hMBL). Analysis under nonreduced conditions showed rMBL A and rMBL B to form higher order oligomers (monomers to hexamers) as observed for hMBL. Functional analysis of the rMBL molecules ability to bind sugars measured by ELISA, upon extensive dialysis of the culture medium, indicated that both rMBL A and rMBL B bind to mannan. Functional studies related to complement activation are currently being investigated. The estimated expression level of rMBL A and rMBL B (~0.3mg/L) indicates that the yeast system may be a better expression system of rMBL than the insect cell system (~0.2mg/L). Research supported by NIH grant HL073804