Purification of TK and crystallisation using hanging-drop method

The purification strategy involved resuspending E.coli pQR 700 cells containing transketolase in 50 mM Tris-HCl, pH 7.5,1 mM EDTA, (3-

mercaptoethanol, PMSF and benzamidine (Boistelle and Astier, 1988) (Section 12.2.1). The resuspended cells were homogenised at 1100 bar with 4 passes through the Lab 40 homogeniser (Section 12.2.1). The homogenised TK extract was clarified in Eppendorf tubes using a Microcentaur centrifuge (Section 12.2.2). Protamine sulphate was added to the clarified TK extract at a concentration o f 0.01 % (w/v) to remove any DNA present in the preparation (Section 12.2.4.2). The removal o f DNA

Appendix

was essential since it might interfere with the binding o f TK onto an ion-exchange column later on in the purification procedure.

A 50% (w/v) ammonium sulphate precipitation (Section 12.2.4.2) was used to remove any contaminating proteins in the TK extract. Dialysis o f TK against a larger volume (2 litres) o f Buffer A (Section 12.2.1) through a semipermeable membrane was to remove the sulphate ions that were inhibitory to the transketolase (Littlechild

et a l, 1995). Sulphate and phosphate ions were found to inhibit transketolase approximately 50% between concentrations o f 0.01 and 0.02 M (Littlechild et a l,

1995). Dialysing the TK twice minimised inhibition by reducing the sulphate concentration down to 3.3 pM.

The dialysed transketolase was then loaded onto an FPLC ion-exchange column using a Fast-Flow Q (FFQ) sepharose as a support (Section 12.2.4.2). The FFQ Sepharose ion exchangers are highly cross-linked agarose beads o f high chemical and physical stability (Ion Exchange Chromatography Principles and Methods, Pharmacia Biotech). These 90 pm beads have a triethyl aminoethyl

functional which remain charged over a wide pH range. A pH o f 7.5, greater than the pi o f transketolase, was chosen so that TK would exist in its negatively-charged state for binding onto the ion-exchanger.

TK was eluted o ff the column by applying a gradient o f salt (NaCl) from 0-1 M. The counter ion Cl ' competes with TK for binding to the ion exchanger and at a specific concentration displaces TK from the column. The total activity o f TK was assayed at each stage using the enzyme linked assay system (Section 12.2.9.1).

Appendix

T able 16 of Escherichia coli transketolase:

Stage Volume (m L) Am t. p rotein

(mg)

Total Units (U*) A ctivity (U^.mg-') % Yield P urification F a c to r Crude extract 10 180 2600 14.4 100 1.00 protamine sulphate precipitation 9.5 142.5 3990 28.0 153 1.94 50-75% (w/v) ammonium sulphate precipitation 9.5 38 950 25 36.5 1.74 Dialysis 20 20 800 40 30.8 2.78 ion-exchange 10 6.28 680 108 26.2 7.50

* nmole o f NADH utilised per minute at 308K, pH 7.6 in the linked assay system.

Percentage yield represents the total units o f enzyme after each stage divided by the total units in the crude extract

Purification Factor represents the specific activity after each stage divided by the specific activity in the crude extract

Appendix

93 kD

66 kD

45 kD

Gel 3; A 12.5% SDS-PAGE gel of the purification of TK. Lane 1 is the low

molecular weight markers. Lane 2 is clarified crude TK extract. Lane 3 is TK treated with protamine sulphate. Lane 4 is TK treated with 50 %-75 % (w/v) ammonium sulphate. Lane 5 is TK after dialysis. Lanes 6 and 7 are TK after FFQ Sepharose column chromatography.

Using this strategy, transketolase was purified to a factor o f 7.5 with a final yield of 26.2 % as recorded in Table 16. The TK after ion-exchange chromatography appeared quite pure (Gel 3) and was used for crystallisation according to the

Hanging-drop vapour diffusion technique (Section 2.4.4 ). Crystals were grown in 50 % (w/v) ammonium sulphate containing 2 mM Thiamine Diphosphate (T P P ), 9 mM CaCb in 50mM PIPES buffer at pH 6.4. These crystals had a needle-like morphology characteristic o f transketolase as shown by Littlechild (1995).

12.3.3 Batch purification of TK

Appendix

no technical difficulties caused by swelling or shrinking o f o f ion exchanger beads. Most o f the TK were bound to DE 52 at low salt concentration (20 mM Tris-HCl, pH 7.6) and were eluted at 200 mM salt concentration (Figure 47). The percentage yield o f TK recovery according to Table 17 was very high (98.8%), however TK was only purified to a factor o f 2.7. Gel 4 (lane 3) showed that this purification method removed the contaminating proteins o f higher molecular weights in the preparation. The purification factors obtained using the batch method and FFQ Sepharose column were the same (PF = 2.7). Comparing Tables 16 and 17, the percentage yield using the batch method was better than the FFQ column. The batch method was also much simpler and cheaper to operate at a larger scale.

c 140 g I 120 “ 100

i

o c 3

I

300 0 100 200 400 500

Ionic strength of NaCi (mM)

Figure 47: Elution profile o f TK using DE52 beads. □ , total units o f TK bound onto the DE52 beads by assaying the supernatant after the beads were

washed in low salt buffer. # , total units o f the eluted TK after each salt concentration.

Appendix

Table 17; Purification using batch separation method Stage Volume (mL) Amt. protein (mg) Total Units(U*) Activity (U*.mg-*) % Yield Purification F actor Cr ude extract 1 20 130 6.5 100 Batch separation 1.8 7.2 126 17.5 98.8 2.7

* inmole of NADH utilised per minute at 308 K, pH 7.6 in the linked assay system.

97 kD

66 kD

45 kD

Gel 4; A 12.5 % SDS-PAGE showing the batch separation purification. Lane 1 shows the low molecular markers. Lane 2 shows clarified crude TK. Lane 3 is the TK purified using the batch separation method.

Appendix

In document The effect of crystal form on the catalytic and mechanical stability of cross-linked enzyme crystals (CLECs) (Page 179-185)