The Sulfolobus acidocaldarius S-layer is composed of two main proteins: SlaA, which forms the ordered structure of the S-layer matrix, and SlaB, which supports and anchors the S-layer in the tetraether lipid membrane. Large batch-scale methods using starting culture volumes of 20-70 L have been developed for the isolation of the SlaA protein from the rest of the cell envelope. These methods rely on the thermotolerance of SlaA and its high resistance to detergents. SlaB, however, remains strongly associated in a large molecular weight aggregate with other cell envelope components, even after removal of the remaining cellular contents.Here we demonstrate a protocol for benchtop-scale (1-2 L) purification of S-layer proteins. Using this protocol, we were also able to identify for the first time the tetraether lipids strongly attached to SlaB.

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