I am trying to express the human MD2 (the cofactor of TLR4) in BL21(DE3) in the vector pET41a. There was no expression band of SDS-PAGE gel at all. This is not the first time we recruited the pET41a and BL21(DE3). They worked pretty well for expressing the other two proteins in our lab (yielding about 100 mg per liter LB medium culture).
The plasmid was able to transformed and selected in kanamycin agar plate. But the plasmid extraction yield was quite low (less than 50 ng/uL from 1 mL LB mini-prep of DH5a). I suspected that maybe there was some Insertion Sequence coincidently. But I tried to codon optimized twice and neither worked.
Any ideas or comments will be appreciated!
Is the sequence you uploaded actual experimental sequencing data of the whole plasmid or is it just what the hypothetical sequence is?
Also, have you tried Western blotting and checking the membrane fractions pre and post IPTG addition for expression? Since MD2/ly96 supposedly binds a component critical to the cell membrane integrity of E. coli (lipid A) I would expect it to localize to the membrane fractions, and probably be extremely detrimental to E. coli cell growth. Though it would be weird if it was also inhibiting the growth of DH5a where it shouldn't be expressed, unless the low yield was from a coincidentally bad prep or there's a cryptic promoter upstream of the T7 promoter.
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