User:Kinkreet/Protein Science/Purification of Fap1-nr2

Streptococcus parasanguis is a member of sanguis streptococci, a group of Gram+ bacillus that are normally found in the mouth. It can adhere to the pellicle (the thin layer of proteins that binds salivary glycoproteins, and protects the tooth against acids and also prevents calcium phosphate deposition) that lies on the dental surfaces, and initiate biofilm (known as dental plaque) formation. The S. parasanguis can subsequently be bound by other microorganisms that causes caries, periodontitis, and other oral diseases.

The adhesive properties of S. parasanguis lies with the presence of peritrichous fimbria, which acts as a scaffold for adhesin to act between the bacteria and the host cell. This was tested in vitro using S. parasanguis FW213 attaching to saliva-coated hydroxyapatite (SHA), an in vitro model of the tooth surface. When fap1 (fimbria-associated protein 1), a 7659-nucleotide gene encoding for an adhesin, was cloned and inactivated by insertional mutation, it led to null expression of the fap1 adhesin, and prevented fimbria formation; thus mutant S. parasanguis which lacks the fap1 gene was unable to form biofilms.

During dental cleaning and surgeries, some of the bacteria may gain entry into the bloodstream and cause plaques to be established else where, such as the heart valves, where it can cause endocarditis, which is a type of inflammation. From UniProt, the length of the amino acid sequence is known to be 2587 residues in length (~200 kDa in molecular weight ), which is further modified post-translationally, predominantly by being glycosylated in the cytosol.

Using the nucleotide sequence to predict the protein sequence and function, Fap1 appears to have a unusually large signal peptide (50 amino acid residues in length), a sorting signal destining towards the cell wall; but the majority (~80%) of the coding region is taken up by two repeat regions (I and II). Repeat region I have 28 dipeptide repeats of (E/V/I)S, whereas repeat region II have ~1000 dipeptide repeats of the same type. There are also two non-repeating regions near the N-terminus, Fap1-nr1 and Fap1-nr2. Fap1-nr1 consists of 52 residues and Fap1-nr2 contains 331 amino acids residues (residues 175-505).

We have characterized Fap1-nr2, with the aim of aiding our understanding of the functions of the two non-repeating fragments, specifically their effects on adhesion.

Cloning
The DNA coding sequence of the Fap1-nr2 fragment was cloned into a vector and expressed in BL21 (DE3) competent E. coli. Bl21 competent cells are designed for high level of protein expression, using the T7 RNA polymerase promoter expression systems; they also lack OmpT and Lon proteases, which, if present, may degrade recombinant proteins. The gene which encodes for endonuclease I (endA), is also inactivated, to ensure the plasmid DNA is not degraded.

Additionally, the vector provides the sequence encoding a 6xHis at what will be the N-terminus of the peptide, to aid in purification using affinity chromatography.

The transfected cells were grown in LB broth overnight, shaking at 37°C, and selected using 50μg/ml. The culture was diluted 10 times, and grown until an O.D.600 of 0.6 is reached. Four hours before harvesting, the bacteria are treated with IPTG to induce the expression of the insert by binding the lac repressor. The bacteria are then harvested by centrifugation.

The harvested cells were resuspended in 25ml binding buffer (50mM NaPO4 buffer, pH 8.0, 0.3M NaCl, 1mM imidazole) and lysed using a French press (16000 rpm, 20 minutes). The supernatant, containing the majority of the Fap1-nr2 was used for subsequent purification.

Purification
50% Ni2+-agarose resin was used to bind to the 6xHis tags of the Fap1-nr2 fragment. The column was conditioned using deionized water and binding buffer, before 20ml of supernatant was added to the column. It is then washed with 10ml of binding buffer, and eluted with 10ml of elution buffer (50mM NaPO4 buffer, pH 8.0, 0.3M NaCl, 60mM imidazole). 10 1ml fractions were collected.

Bradford assay
Bradford assay was used to determine the approximate concentration of proteins in each sample. Albumin (0.05 - 1.00 mg/ml) was used to determine a standard curve. Aliquots from the 1ml fractions obtained from column chromatography was added to Bradford reagents, and left for 10 minutes to equilibrate. The O.D. at 595nm was measured and used as an indication of protein concentration of each fraction.

SDS-PAGE
The first five fractions (those that contained the most proteins) was loaded onto 12% SDS-polyacrylamide gels. 10μl of each fraction was loaded and ran at 200V for 1 hour. Protein bands was visualized by incubating with Instant Blue stain for 1 hour, and then washing with water for 1 hour to reduce background staining.

Trypsin digestion
The purified Fap1-nr2 sample is diluted to 0.2mg/ml with digestion buffer (200mM Tris-HCl pH 7.7 and 4 mM CaCl2). Trypsin (at a ratio of 1:50 relative to Fap1-nr2) is added to the sample for 0, 1, 2, 5, 10, 20, 30 and 60 minutes. The sample is incubated at 37°C, for the allotted time, after which the digetsion is stopped using stop solution (25mM EDTA, 2mM PMSF, 2x SDS loading buffer).

SDS-PAGE
The digested samples stopped at different time points are ran on a 12% SDS-polyacryamide gel at 200V for 50 minutes, and proteins bands visualized using Instant Blue stains, as before.

Results
Fragments