TABLE 1.
Binding of 125I-labeled type I and IV collagen to the S-layer proteins and formation of a crystalline layer by the proteins
| Test protein | Binding (%)a of
|
Formation of crystalline layerb | |||
|---|---|---|---|---|---|
|
125I-collagen type I
|
125I-collagen type IV
|
||||
| Mean | Range | Mean | Range | ||
| Native or His-tag fusion S-layer proteinc | |||||
| CbsA | 120 | 81–145 | 122 | 66–162 | + |
| His-CbsA | 100 | 100 | + | ||
| SlpA | <1 | 0–1 | <1 | 0–1 | + |
| His-SlpA | <1 | 0–1 | <1 | 0–1 | + |
| His-CbsB | <1 | 0–1 | <1 | 0–1 | + |
| His-SlpnB | 5 | 4–6 | 2 | 0–4 | + |
| Hybrid His-tag fusion S-layer protein | |||||
| His-CbsA1–212/SlpA208–413 | <1 | 0–1 | 1 | 0–2 | − |
| His-SlpA1–207/CbsA213–410 | <1 | 0–1 | <1 | 0–1 | − |
| His-CbsA1–287/SlpA290–413 | 61 | 22–111 | 72 | 40–92 | + |
| His-SlpA1–289/CbsA288–410 | <1 | 0–1 | <1 | 0–1 | + |
| His-CbsA1–28/SlpnB20–409 | <1 | 0–1 | <1 | 0–1 | + |
| His-SlpnB1–19/CbsA29–410 | 105 | 87–153 | 97 | 74–117 | + |
| His-CbsA1–81/SlpnB73–409 | <1 | 0 | <1 | 0–1 | − |
| His-SlpnB1–72/CbsA82–410 | 78 | 27–131 | 61 | 44–86 | + |
| His-CbsA1–194/SlpnB187–409 | <1 | 0–1 | <1 | 0–1 | + |
| His-SlpnB1–186/CbsA195–410 | <1 | 0–1 | <1 | 0–1 | + |
| His-CbsA1–212/SlpnB205–409 | <1 | 0–1 | <1 | 0–1 | + |
| His-SlpnB1–204/CbsA213–410 | <1 | 0–1 | <1 | 0–1 | + |
| His-CbsA1–250/SlpnB250–409 | <1 | 0–1 | <1 | 0–1 | − |
| His-SlpnB1–249/CbsA251–410 | <1 | 0–1 | <1 | 0–1 | − |
| His-CbsA1–287/SlpnB287–409 | 54 | 39–96 | 73 | 47–98 | + |
| His-SlpnB1–286/CbsA288–410 | 2 | 0–3 | 4 | 4–5 | + |
| Polypeptide | |||||
| His-CbsA1–212 | 2 | 0–2 | <1 | 0–1 | − |
| His-CbsA1–250 | 1 | 0–2 | 1 | 1–2 | − |
| His-CbsA1–287 | 77 | 65–83 | 73 | 27–77 | + |
| His-CbsA42–287 | 4 | 2–5 | <1 | 0–1 | − |
| His-CbsA288–410 | 2 | 2–3 | <1 | 0–1 | − |
| Mutated CbsA protein | |||||
| His-CbsA Δ22–26 | 2 | 0–2 | 8 | 5–15 | + |
| His-CbsA Δ91–96 | 22 | 20–24 | 27 | 19–33 | + |
| His-CbsA NNN14INL | 88 | 50–131 | 74 | 32–100 | + |
| His-CbsA F19S | 83 | 53–109 | 80 | 52–109 | + |
| His-CbsA D130N | 56 | 40–68 | 64 | 39–78 | + |
| His-CbsA N226A | 33 | 24–44 | 34 | 22–40 | + |
| His-CbsA KSDV257TANN | <1 | 0–1 | <1 | 0–1 | + |
| His-CbsA K257T | 91 | 80–102 | 92 | 83–99 | + |
| His-CbsA S258A | 10 | 6–13 | 9 | 4–13 | + |
| His-CbsA D259N | 77 | 63–87 | 87 | 80–98 | + |
| His-CbsA V260N | <1 | 0–1 | 1 | 0–1 | + |
| His-CbsA TA264SK | 52 | 44–64 | 49 | 36–68 | + |
| His-CbsA P268A | 51 | 34–66 | 44 | 36–54 | + |
| Control | |||||
| BSA | <1 | 0 | |||
a
Results are mean values from at least six independent assays; the range gives the lowest and the highest value obtained in the assays. The binding value shown by His-CbsA was assigned as 100% in each individual test.
b
Formation of the S-layer was determined by transmission electron microscopy of the purified proteins negatively stained with phosphotungstate acid.
c
“Native” denotes to the S-layer extracted from Lactobacillus cells. His-tag fusion proteins were isolated from E. coli cells.