Properties of salivary mucins: MUCSB protein expression by isolation of C-terminal constructs in the cystine knot motif

Abstract

The present study of salivary mucin MUC5B has sought to isolate and evaluate the properties of peptide constructs derived from within the cystine-knot motif. Three peptide constructs of different lengths were designed from the C-terminal sequence of MUC5B and each construct incorporated restriction sites for the endonucleases BamHI, at their 5' end, and Xhol at their 3' end. Amplification was accomplished using RT-PCR, with cDNA for the RT reaction derived from RNA isolated from frozen human submandibular glands. Constructs were cloned in to E. coli using a TOPO vector and cultured in order to isolate plasmid inserts that were then ligated into a pGEX-5X-2 vector. BL21 E. coli were utilized as host cells, using the pGEX-5X-2 vector to transform the construct plasmids. Protein expression was then induced in the host cells using IPTG and the GST fusion-protein product collected via centrifugation of the sonicated cell pellets. Fusion proteins were then isolated using Sepharose 4B affinity column purification. Isolates were finally dialyzed against deionized water and lyophilized; the lyophilate was then intended for immunization of rabbits. Fusion protein isolation required extensive optimization, including the reduction of sonication cycles and the addition of protease inhibitors in order to reduce the action of host cell enzymes on the protein product. Attempts to cleave constructs from their GST fusion partners were unsuccessful due to the possible effects of intramolecular folding during expression that may have blocked the enzyme recognition site. Denaturants were employed in order to improve enzyme-protein interaction, however, colorimetric assays revealed that even suggested levels of these chemicals significantly reduced the activity of the cleavage enzyme.