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The G48V “extended proteinase” crude protein sample was purified further on Ion exchange on

DEAE-Cellulose (5.1.5).

Fig. 5.30: SDS-PAGE analysis of protein sample G48V extended proteinase” by DEAE- Cellulose chromatography. Samples were subjected to 20% gel concentration and polymerised gel was stained with coomassie blue (pH 5.6).

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A

4

Lane 1 : Biorad blue™ marker (prestained protein standards)

Lane 2; Crude G48V “4833” ; before DEAE-Cellulose chromatography Lane 3: Crude 0 4 8 V “4833” ; after DEAE-Cellulose chromatography

From the gel shown in figure 5.30 only the larger molecular weight proteins (40 kDa and above) bound to the positively charged ions o f the gel. This indicates they contain negatively charged moieties. The protein bands between 13 and 18 kDa protein eluted from the column together. Since there was no separation between 13-18 kDa proteins, another experiment for purifying the crude G48V “extended proteinase” by gel chromatography column was attempted.

Essentially, the experiment was repeated on the Ion exchange Sp- sepharose column in order to find out whether the protein samples behaved differently.

Fig. 5.31: SDS-PAGE analysis of protein sample G48V “extended proteinase” by SP- sepharose chromatography. Samples were subjected to 20% gel concentration and polymerised gel was stained with Coomassie blue.

1 2 3 KDa

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Lane 1 : Biorad blue^"^ marker (prestained protein standards)

Lane 2 : Crude G48V “4833” ; before SP-sepharose chromatography Lane 3: Crude G48V “4833”; after SP-sepharose chromatography

The result of SDS-PAGE analysis figure 5.31 shows that only a 14 kDa protein o f the applied material bound to the SP-sepharose column.

The bound proteins were eluted in 14 ml fractions with a salt concentration gradient (0.1 M NaCl to 1 M NaCl). 10 fractions o f 14ml were collected. Fractions eluted with different salt concentrations 0.2 M NaCl-0.5 M NaCl were analysed on SDS-PAGE and as shown in figure 5.32 the 14 kDa protein eluted o ff the column with 0.4 M NaCl.

Fig. 5.32: SDS-PAGE analysis of elution fractions of G48V “extended proteinase ”by SP- sepharose chrom atography. The purification o f 14kDa protein was clearly observed.

KDa

14

Lane 1 : Biorad blue™ marker (prestained protein standards) Lane 2: Protein sample eluted with 0.2 M NaCl.

Lane 3: Protein sample eluted with 0.3 M NaCl. Lane 4: Protein sample eluted with 0.4 M NaCl. Lane 5: Protein sample eluted with 0.5 M NaCl.

5.1.17: Properties of the purified G48V “extended proteinase”

The molecular weight o f the 14 kDa protein was determined using a mass spectrometer by ULIRS mass spectrometry service and estimated at 14,367 (figure 5.33).

Fig. 5.33 : Graph Showing the molecular weight of the G48V “4833” protein sample fraction (14 kDa) hy SP-sepharose protein using mass spectrophotometer.

7X83

F ile ;9 7 T 0 F 3 3 3 8 I d e n t : 1 5 H 0 (1 . 7rPK IH 7, 3 , 7 , 0 . 2 5 » . 0 ■ 0,"5CT 00%, F. T P Acq : lS - D EC -1 9 g 7 1 1 ;3 Ü T 5 7 C a l:C A » |

TOFSPEC LDI+ Parents

File Text:Modefled G 48V proteinase (1) Matrix : DHAP/DAC (LINEAR Number of shots averaged

100*1 _2.0E3 98. 96. 94- 92. 90. 88. 8 6. 84. 82. 80. 78 76. 74. 6130 10759 12158 _{|16573 18663 20392 22465 24030}

éo 'o ô i o 6 o b 1 2 6 0 0 1 4 6 0 0 16 6 0 0 i'8 (io'o 2bdioo 2 2 6 0 0 2 4 6 0 0

.2.0E3 .1.9E3 .1.9E3 .1 . 8E3 .1 8E3 . 1 .8E3 . 1 .7E3 . 1 .7E3 . 1 ,6E3 .1 6E3 -1 6E3 . 1 .5E3 _1.5E3

Fig. 5.34: Graph showing the molecular weight of lysozyme using mass spectrophotom eter

F ile :9 7 T O F S 2 3 I d e n t : 1 S M 0 [1 ,7 ) PKD(7 , 3 , TTDT25«70 .0 ; 50 . 00%, F, Tl SMO( 1 , 7 T PKD(7 , 3 , TT'O . 25%, 0 . 0 , 50 . TOFSPEC L D I. P a r e n t s

HABA (LINEAR Number of shots averaged 100. Laser Energy 4:120 A»

14346 ^1.3E3

File Text :Lysozyme Matrix:

100» 95 90 85 80 75 70 65 60 55 50 45 40: 28797 33654 37113 1 .2E3 1 .2E3 1.1E3 1.0E3 9 ,7E2 9 . 1E2 8.4E2 7 .8E2 7.1E2 6.5E2 5.8E2 5.2E2

5o'o6 i o 6 o b 1 5 6 0 6 2 0 6 0 0 ' 2 5 6 0 0 3 od)ob ' ' 3 5 (1)00 ' 4 0 6 0 0 ' 4 5 6 0 0 ' 50A 00 m /z

The activity o f the protein was also measured at 9% using peptide cleavage HPLC. As this protein had a molecular mass very close to that o f lysozyme (figures 5.33 and 5.34), further tests were carried out on this protein to ensure that it was not a lysozyme contaminant left behind following cell lysis. Therefore, it was decided to compare the molecular weight o f lysozyme and the 14 kDa protein eluted with 0.4 M NaCl on SDS-PAGE gel (shown in figure 5.35).

Fig. 5.35: Comparison of the elution fraction of the G48V extended proteinase” by SP- sepharose with lysozyme on SDS-PAGE.

Samples were subjected to 20% gel concentration and polymerised gel was stained with Coomassie blue.

KDa

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Lane 1: Biorad blue™ marker (prestained protein .77

standards)

Lane 2: Lysozyme

Lane 3: The 14 kDa protein eluted o ff the SP-sepharose column in 0.4 M NaCl. Lane 4: Mixture o f the above two samples.

Further analysis was carried out on western immunoblot. The 14 kDa protein was transferred to a

nitrocellulose membrane which was probed with anti HIV-1 proteinase and anti reverse transcriptase antibodies (2.3.4). As shown in figures 5.36 A and 5.36 B, the results show presence o f purified 14 kDa protein with both antibodies, but lysozyme is not recognized by the RT antibody.

Fig 5.36: Western blot analysis of purified 14 kDa protein by Sp-sepharose ion exchange chromatography and lysozyme.

Lysozyme has low affinity for the PR antibody.