5. ANÁLISIS INTERNO
6.1. MATRIZ DE EVALUACIÓN DE LOS FACTORES EXTERNOS (MATRIZ
(contained in first peak)
2
2.0
2.5
Volume (ml)
7. 16: Elution Profile of Size Exclusion Standards
The elution profile is created by continuously monitoring the A280. The absorbance scale is approximately 0-0 . 5 absorbance units.
7-1 78 1.9 1.8 1.7 - '6, 1.6 ";r; � ai 1.5 'S (.) � 1.4 0 E
�
1.3 1.2 1.1 1 0.95 1 1 .05 1.1 1.15 1 .2 1.25 1.3 1.35retention time (minutes)
7. 17: Calibration Curve Constructed From Elution Profile
7-1 79
hydrated) HTP hydroxyapatite resin (Biorad) pre-equilibrated in initial buffer. After 1 0- 1 5 min to allow binding, the mixture was centrifuged at 3 000 x G for 5 min in a Sorvall SM24 rotor. The supernatant was removed and kept for analysis. The procedure was repeated adding 1 mI of each increasing concentration phosphate buffer each time. The supernatants were analysed for content by SDS-P AGE and IEF gels with activity staining. Unfortunately no activity remained after this process. In addition, SDS-P AGE showed that none of the proteins appeared to bind to the resin. All Coomassie stained bands appeared in the supernatant from the 1 0 mM phosphate buffer wash.
7.2.9 Gel Purification
Because the initial successful purification of the novel protein had not been able to be repeated, and no other methods tried had worked, the next step was to purify the protein from an SDS gel and try to get some sequence information. With enough sequence information to 1 ) identify the protein as novel, and 2) place the protein in a known family of proteins, it was hoped that this information could be used to design oligonucIeotides to screen a sheep liver cDNA library, and clone and express this protein for further characterisation.
Initially, because the molecular weight, isoelectric point, organism and tissue of origin, and an activity of the protein had been established, this information was entered into the SwissProt 2D electrophoresis database to see if any known protein matched these characteristics. This search yielded a number of potential matches from the alcohol dehydrogenase family with basic isoelectric points and similar subunit molecular weights including the class IV retinol dehydrogenase. However, these proteins are all dimeric, while it appears that the new protein is monomeric in its native state. So all available evidence still indicated that the protein was novel, and only a positive sequence identification would confirm this.
To purify the protein from SDS-PAGE, a 1 . 5 mm thick, 1 2 % (w/v) 37.5 : 1
acrylamide:bisacrylamide SDS gel with one large lane, and one lane for molecular weight standards was cast (section 2.2. 1 0) and run as shown (Figure 7. 1 8). After the gel had run, it was stained with Coomassie blue R250, as described in Materials and Methods. The band corresponding to 40-45 kDa was excised using a scalpel, and placed into dialysis tubing with 2-3 ml of 50 mM Tris-HCl pH 7.4 containing 0. 1 % (w/v) SDS. The dialysis tubing containing the protein was placed into a horizontal electrophoresis tank,
7-1 80
filled with the same buffer. The protein was eluted from the gel by applying a 5 0 mA current for approximately 48 hours. The current was reversed for 5 - 1 0 min to remove any protein which may have adhered to the dialysis tubing. The protein-containing solution was then aspirated from the tube and concentrated under vacuum. An analytical SDS-P AGE gel showed that the protein was 95- 1 00 % pure (Figure 7. 1 9) .
66 kDa 45 kDa 3 6 kDa 29 kDa 24 kDa 20 kDa 1 4.2 kDa - - - - - - -
SD S-7 Ladder Partially Purified Mixture
of Interest
7.18:
Schematic
of Gel Purification
The SDS-PAGE is run as usual (see Materials and Methods). After staining, the band of correct size is identified using the protein ladder, and gently excised from the gel.
The first analyses carried out on the purified protein were another attempt at N-terminal sequencing and an amino acid analysis. To ensure that previous sequencing efforts had not failed due to insufficient quantity of protein, N-terminal sequencing was repeate,d. This was done by running a sample of the purified protein on a 1 5 % (w/v) 3 7 .5 : 1 acrylamide: bisacrylamide SDS-gel. The gel was then electrotransferred onto PVDF membrane, as described in Materials and Methods, and sequenced. No sequence was obtained, which supported our initial hypothesis that the N-terminus was blocked. The amino acid analysis yielded the composition of amino acids of this protein (Table 7. 1 ). From the resulting chromatogram of total amounts of each amino acid, the actual composition was calculated using the following formula:
Moles of amino acid Molecular weight of protein
x
7-1 8 1
where the total moles in the sample was 1 2 1 moles, and the two protein molecular weights used were 42 kDa and 45 kDa for two different calculations (Table 7 . 1 ) .
Table 7. 1 : Amino Acid of Unknown Protein
Amino Molecular Residues % of Total Residues % of Total
Acid Ala 79 3 9 1 0 . 5 42 10.5 Glv 75 85 22.9 9 1 22.8 Val 1 17 32 8.6 34 8.5 Leu 1 3 1 25 6.7 27 6.8 Ile 1 3 1 1 3 3.5 14 3 . 5 1 47 18 4.9 19 4.8 175 9 2.4 10 2 . 5 Asx 132 24 6.5 26 6.5 Glx 1 46 28 7. 5 30 7.5 His 156 6 1 .6 6 1 . 5 Pro 1 15 17 4.6 1 9 4.8 Thr 1 19 1 9 5. 1 20 5.0 Ser 105 37 10.0 40 1 0.0 122 4 1 . 1 5 1 . 3 Met 149 4 1 . 1 4 1 . 0 Phe 166 9 2.4 1 0 2 . 5 1 82 2 0.5 2 0.5 204 0 0 0 0
The resulting ammo acid composition was submitted to two database searching programs, ExP ASy and PROPSEARCH, for matching with any other known protein. No close matches were found. The five closest matches made were: 1 ) fruit fly protein kinase sgg39, 2) hypothetical protein UL 1 1 2, 3 ) protein kinase shaggy, zygotic, 4) fruit fly zeste-white 3 -A, and 5) radish acetoacetyl-CoA-thiolase (all using 42 kDa molecular weight), and 1 ) sporozoite surface antigen, 2) procollagen EMFl -a, 3 ) neuroblast differentiation associated protein, 4) human AHNAK protein fragment, and 5) pecanex protein fragment (all using 45 kDa molecular weight). None of these have characteristics similar to the novel protein, and the closest match was less than 68 %
reliable according to the program. Again, this search supports that this protein is a previously unidentified protein. However, to classify the protein as an alcohol dehydrogenase and confirm its uniqueness, some positive sequence information must be obtained.
7- 1 82 LANE: 1 2 3 kDa kDa kDa kDa kDa 20 kDa 1 4.4 kDa