Analysis of the MBP-PRDS L3-4 loop by native PAGE

A sample o f the fusion protein was analysed by a 6% native PAGE. However, the protein remained at the top o f gel even in the presence o f lOmM DTT, indicating a high level o f aggregation.

3.6.6 Dimérisation studies

The ability o f PRDS to form dimers with itself and with ROM-1, a non-glycosylated related protein, has been documented (40). Since the loop region is the largest part o f the protein it was thought that many o f the interactions for dimerization occurred through this region. In part this was later verified when mutation o f one o f the cysteine

C hapter 3 C haracterisation o f the P R D S L 3 -4 lum en lo o p and m utant varian ts kDa 1 2 3 4 5 6 66 ► 45 ► 36 ► 2 9 ^

P iG. 3.7 Expression of the M BP-PRDS L3-4 loop

Samples were analysed on a 12% SDS PAGE stained with Coomassie Blue. Lanes: 1, Dalton protein molecular mass marker; 2, MBP protein only ( 15pg); 3, whole cell lysate from bacteria transformed with MBP-PRDS L3-4 loop region uninduced; 4, whole cell lysate from bacteria transformed with M BP-PRDS L3-4 loop region induced with IPTG; 5, soluble fraction after sonication of the fusion protein; 6, insoluble fraction after sonication of the fusion protein from the J M 101 bacterial strain. MBP; maltose binding protein.

kDa 1 2 3 66 ^ 45 ► 3 6 ^ 29 ► 20 ►

Fig. 3.8 Purification o f M BP and M BP-PRDS L3-4 loop

Samples were analysed on a 12% SDS PAGE stained with Coomassie Blue. Lanes: 1, Dalton protein molecular mass marker; 2, MBP isolated by

amylose affinity chromatography (lOpg); 3, M BP-PRDS L3-4 loop isolated by amylose affinity chromatography (lOpg) from the JM lO l bacterial srain.

42kDa MBP

14

lOkDa PRDS L3-4 loop

Fig. 3.9 Cleavage of the MBP-PRDS L3-4 loop fusion protein

Samples from the trypsin digest was analysed on a 12% SDS PAGE and stained with Coomassie Blue Lanes; 1, Dalton protein molecular mass marker; 2, limited proteolysis of the M BP-PRDS L3-4 loop

3 4 5 6 7 region. kDa ₁ 205 ► 116 ► 97 ► 66 ► Dimer band 45 ►

Fig. 3.10 Dimérisation o f M BP-PRDS L3-4 loop and mutant

variants

Samples after air oxidation were analysed on a 6% SDS PAGE and stained with Coomassie Blue. Lanes; 1, High molecular mass protein marker; 2, MBP protein only; 3, MBP-PRDS L3-4 protein; 4, MBP- L185P protein; 5, MBP-R172W protein; 6, M BP-R172G protein; 7,

C hapter 3 C haracterisation o f the P R D S L 3 -4 lum en lo o p and m utant varian ts

C T f l C C C G G R C

190

C T A C C T G G A C W ildtype

F ig . 3.11 L185P mutant

Electropherogram of the mutated DNA sequence at Leu 185 changing leucine to proline.

R T f l C T G C G G C165Y

130

A T G C T G C G G Wildtype

F ig . 3.12 C165Y mutant

Electropherogram of the mutated DNA sequence at Cys 165, changing cysteine to tyrosine.

G T T T T G G G G R 1 7 2 G 18(

G T T T T G G G G W ildtype

F ig.3.13 R172G m utant

Electropherogram of the mutated DNA sequence at position R172 changing arginine to glycine.

G G T T T T T G G G

HO R172W

G G T T T T C G G G W ildtype

F ig . 3.14 R172W mutant

Electropherogram of the mutated DNA sequence at position R172 changing arginine to tryptophan.

C hapter 3 C h aracterisation o f the P R D S L 3 -4 lum en lo o p and m utant varian ts

F ig . 3.15 In vitro translation o f wildtype and mutant

constructs under reducing conditions

Samples from in vitro translation were analysed on a 10% SDS PAGE in the presence of p-mercaptoethanol and a false colour phosphoimage taken. Lanes: 1, Dalton protein molecular mass marker; 2, His-human; 3, His-C165Y; 4, His-R172W; 5, His- R172G; 6, His-L185P (Courtesy of Jonathan Wrigley)

1

m

F ig . 3.16 In vitro translation o f wildtype and m utant constructs under

non-reducing conditions

Samples were analysed after in vitro translation on a 10% SDS PAGE and a false colour phosphoimage taken. Lanes: 1, His-human; 2, H is-C l65Y; 3, His-R172W; 4, His-R172G; 5 His-L185P, under non-reducing

conditions. Non-reducing conditions were obtained by the inclusion of a final concentration of 2mM GSSG in the reaction, and samples from these expressions were prepared in the presence of 9mM lodoacetamide.

Numbers to the left of the image represent the protein molecular mass markers (Courtesy of Jonathan W rigley).

kDa 45 ► 36 ^ 29 ► 24 ► 42kDa MBP 17kD aL185P 1 2

Fig.3.17 Cleavage o f the M BP-L185P fusion protein

Samples from limited proteolysis of the L185P-PRDS mutant protein were analysed on a 12% SDS PAGE and stained with Coomassie Blue. Lanes: 1, Dalton protein molecular mass marker; 2, cleavage of M BP-L185P, 2 bands observed corresponding to MBP (42kDa) and L185P-L3-4 loop (17kDa).

MBP / Tris 20mM MBP / urea 7M M BP/urea 0 350M 4* -2- X 300 -4- 200 220 240 260 280 300 320 W avelength (nm) Fig. 3.18 CD spectrum o f M BP only

Chapter 3 Characterisation o f the P R D S L 3 -4 lo o p and m utant variants

reducing reagents were removed from the sample o f PRDS fusion protein. MBP was also used as a control since it has already been documented that MBP runs as a

monomer. The fiision protein was left in the absence o f reducing agents for up to 72hr at 4°C and a sample o f the proteins mixed with disruption buffer excluding reducing agents and analysed by SDS PAGE (Fig. 3.10). This revealed a series o f bands; one at -5 9 kDa, another at 120 kDa and a band around 240 kDa. The gel was interpreted as the PRDS existing in a number o f species; monomer, dimer, as well as higher

complexes may be oligomers. As mentioned before the size o f the MBP on its own is 42 kDa and under the same conditions, it still migrated as a single band.

In vitro translation studies were also applied to the human and mouse loops in pET-14b to see whether the loop expressed on its own could form dimers. The samples from in vitro translation were analysed by SDS PAGE (Fig. 3.5). Under non-reducing

conditions, this also revealed two bands, a smaller band between 18-20 kDa and a larger one between 34-36 kDa. This strongly suggests that the products o f in vitro translation existed in both monomeric and dimeric forms. Dimerization was enhanced with the use o f oxidising agents such as glutathione. The conclusion from this part o f the study is in agreement with the L3-4 domain o f the protein being important for protein-protein contact.

3.7 Site directed mutagenic studies

It was decided to investigate four PRDS mutations that are known to be involved in retinal degenerations, namely C l65Y (adRP), L185P (digenic RP), R172W/G (macular degeneration) and (pattern dystrophy).

3.7.1 Subcloning of the PRDS L3-4 loop into M 113m pl9

In order to perform site directed mutagenesis, the PRDS L3-4 loop region DNA fragment was subcloned into an M 13m pl9 vector using the Eco RI and H ind III sites. The PRDS L3-4 loop was sequenced completely to ensure that no errors had been introduced into the protein.