1.5 FUNDAMENTOS TEÓRICOS [3], [11], [15]
1.5.3 MICROCONTROLADOR AVR ATEMGA324P [11]
In order to investigate the biological activity of rMRP-14 seen in vivo, the effect of rMRP-14 was studied in vitro using a number of myeloid functional assays. For these assays, rMRP-14 was made in the same way as for the previous in vivo studies, using a bacterial expression system. rMRP-14 was expressed as a His-tagged protein using E.coli BL21 (DE3) plysS transformed with a pET-28a vector into which murine MRP-14 cDNA had been cloned (a gift from R. May). rMRP-14 was produced with an N-terminal Histidine (His) tag, with six consecutive His residues, to aid purification. This results in an increase in its molecular weight from 14 kDa to approximately 16 kDa. Expression of rMRP-14 was induced by addition of IPTG for 4.5 hours. MRP-14 was predominantly expressed as a cytosolic protein (May, 1999), therefore a cytosolic extract was prepared. The bacteria were lysed by freeze/thaw and sonication, then centrifuged to remove insoluble and aggregated material. SDS PAGE analysis of the resultant solution showed rMRP-14 to be a major constituent (Figure 3.1, Lane L).
rMRP-14 was purified by Fast Protein Liquid Chromatography (FPLC) using a Nickel column, to capture the His-tagged protein. Bound rMRP-14 was eluted with an ascending gradient of imidazole (Figure 3.1).
il
O OJII
XI < 1.0 Wash 0 20 40 60 1 I I I I I I I 100 0) B 80 s 60 5 (D 40 c 0) 2 20 0) CL B Fractions kDa M L RI R2 8 9 10 Fractions M 52 53 54 55 56 57 58 59 60 61 62 Fractions pooledFigure 3.1 Nickel purification of rMRP-14 from bacterial lysate
A) The absorbance at 280 nm (black line) of the flow through of the nickel column and the percentage elution buffer (red line; see Material and Methods) during purification of rMRP-14.
B) SDS PAGE analysis of the purification of rMRP-14. rMRP-14 (indicated with an arrow head) was a major constituent of the bacterial cytosolic extract applied to the column (Lane L). rMRP-14 bound well to the Nickel column, indicated by the lack of rMRP-14 in the run through from the column during loading of the sample (Lanes R1 and R2). A large proportion of
contaminating protein was removed from the column with a low stringency wash with 5% elution buffer (Fractions
8
-10). Bound rMRP-14 was eluted with an ascending gradient of imidazole. Fractions containing highconcentrations of rMRP-14 (Fractions 5 5 - 6 1 ) were pooled.
M, Markers; L, Load; R1/2, Run through during column loading; white arrow head, rMRP-14.
Chapter 3: Effect of rMRP-14 in vitro and in vivo
This first purification step was very successful at removing the majority of the contaminating proteins from rMRP-14. Fractions containing high concentrations of rMRP-14 were pooled and further purified using a hydroxy apatite colunrn. Hydroxyapatite resin binds, amongst other macromolecules, calcium binding proteins, and was used to remove any remaining bacterial impurities as well as misfolded non-calcium binding recombinant protein. Correctly folded rMRP-14 was eluted with high concentrations of phosphate, which migrated largely as a single band in SDS PAGE (Figure 3.2). Fractions containing high concentrations of rMRP- 14 were pooled and dialysed into HEPES buffered HBSS, and frozen at - 70 °C in aliquots.
Lipopolysaccharide (EPS) is a component of the Gram negative bacterial cell wall, and as a potent inflammatory mediator, can exert a range of biological effects in vivo, from fever to circulatory failure (“shock”). When preparations are contaminated with EPS, complexes of EPS, protein and phospholipid are produced, and the term endotoxin is used (Henderson and Wilson, 1996). To avoid contaminaton of purified rMRP-14, endotoxin was always removed prior to use of rMRP-14 in in vitro or in vivo assays, using two different methods. Initially, endotoxin was removed using Triton X-114, based on a published method (Aida and Pabst, 1990). The main disadvantage of this procedure is that a small amount of detergent can persist in the purified preparation, which could have unwanted biological effects (Aida and Pabst, 1990). However as a control for this, rMRP-14 was always used in assays alongside a detergent-extracted buffer control.
il
“ I
ü cel î
X) < Wash 0 10 20 30 40 50 60 1 I I I I I I I I I I I I 1.0 _ 100 80 n 60 40 20 0 B kDa M L RI R2 6 7 13 14 27 28 M 29 30 31 32 33 34 35 36 37 46.0 30.0 21.5 14.3 6.5 Fractions pooledFigure 3.2 Hydroxyapatite purification of rMRP-14 from a semi-pure preparation
A) The absorbance at 280 nm (black line) of the flow through of the hydroxyapatite column and the percentage elution buffer (red line; see Material and Methods) during purification. rMRP-14 was already partially purified using a Nickel column.
B) SDS PAGE analysis of the hydroxyapatite column chromatography purification of rMRP-14. rMRP-14 (indicated with a white arrow head) was a major constituent of the solution applied to the column (Lane L). A substantial amount of rMPR-14 was removed from the column with a low stringency wash with 5% elution buffer (Fractions
6
-14), this protein was likely to be mis-folded. Bound rMRP-14 was eluted with 55% elution buffer. Fractions containing high concentrations of rMRP-14 (Fractions 2 8-3 1) were pooled. M, Markers; L, Load; R, Run through during column loading; Fractions6
- 37; arrow head, rMRP-14.Chapter 3: Effect of rMRP-14 in vitro and in vivo
Subsequently, to avoid problems associated with detergent contamination of rMRP-14, a protocol for removing endotoxin using KuttsuClean was used. KuttsuClean has a high affinity for endotoxin and consists of a protein isolated from the amebocytes of the horseshoe crab {Limulus polyphemus) coated on the surface of agarose beads. Purified rMRP-14 was used in assays alongside a buffer control that had been similarly prepared using KuttsuClean. The biological activity of rMRP-14 in the air pouch was retained following endotoxin removal by either of the above protocols ((May, 1999) and data not shown). Residual endotoxin contamination in purified rMRP-14 was measured using the Limulus Amoebocyte Lysate Assay. Following endotoxin removal using the Triton X-114 or KuttsuClean protocols, the level of endotoxin in 50 |ig rMRP-14 was approximately 0.4 ng.