Parámetros que afectan el proceso de electrocoagulación

The following suggestions for future experiments are given in rough order of priority.

3.3.3.1 Analysis of eluent fractions

Because of its high sensitivity, immunoblotting was used to analyze eluent fractions for pl-381 (present at < 20 jLtg.ml”^). À more detailed examination of the standard assay is required, in which the eluent fractions are analyzed by more accurate means:

Protein recoveries

Results obtained by immunoblotting were used to calculate the recovery of pl-381 on the standard assay, and from this result the recoveries of the protein standards were estimated (section 3.3.1.2). More precise protein recovery information is desirable, particularly if the standard assay was to be adapted for use as a preparative technique (section

3.3.3.3). Recovery data for protein standards could be readily obtained by U.V. measurement of eluate fractions and appropriately diluted sample. Interparticulate RNA would have to be removed from purified pl-381 preparations before recovery was measured by this method. This could be achieved by RP-HPLC on the standard assay (section 3.2.2.4), or by

incubation with exogenous nucleases (Burns N.R. et a l . 1992).

pl-381 purity

The absence of background bands on the immunoblot tracks corresponding to eluate fractions gives some indication of pl-381 purity in eluent fractions (eg Figure 3.7, tracks 2, 7 and 10). However, for validation of the assay calibration, where 100 % purity was assumed (section 3.2.3), and for assessment of the possible use of the standard assay as a preparative method (section 3.3.3.3), more detailed purity information is required.

Initial studies were carried out to assess the purity of pl-381 in eluate fractions obtained from miniprep and crude homogenate samples, but no reliable results were obtained. When eluent fraction and control samples were run on a SDS-PAGE gel and protein bands were detected by silver staining, pl-381 failed to stain, due to the small number of cysteine residues (Chuba P.J. and Palchaudhuri S. 1985). An attempt was made to concentrate the protein in eluent fractions by precipitation with trichloroacetic acid, prior to SDS-PAGE and staining with Coomassie Blue. No protein bands were observed on the resulting gels; presumably the proteins failed to precipitate in the presence of the urea and acetonitrile. It is recommended that this second experiment be repeated, but with an alternative concentration procedure (eg dialysis followed by freeze drying).

3.3.3.2 Improvements to the standard assay

During the development of the standard assay, the effect of changing a number of important standard assay parameters was determined (section 3.2.2), but once a satisfactory assay was developed this process was stopped. In order to further improve the standard assay (for example by obtaining base­ line resolution of the pl-381 containing peak in crude homogenates, or by reducing the assay run length) this development process could be continued. The predicted effects of changing a number of other assay parameters are described below, and recommendations for future experiments are made.

The stationary phase

Apart from initial trials with a stationary phase of smaller particle size (section 3.2.2.1), the effect of the stationary phase upon the standard assay performance was not investigated. The adoption of a silica based stationary phase (necessarily at a low pH) is not expected to improve assay performance, since in the analogous prochymosin study solubilized prochymosin was not eluted from either C4 or C» silica based columns (Salt D.E. et al., in preparation), and in this study pl-381 was not recovered from columns at low pH (section 3.2.2.2). A more promising approach would be to test

polymeric stationary phases with different properties to the 30 nm PLRP-S packing used for the standard assay. In particular, the use of wide pore (400 nm) PLRP-S should be investigated. The open structure of this resin is claimed to give significantly improved mass transfer coefficients when compared to its 30 nm counterpart, with high resolution protein separations occurring in under a minute (Lloyd L.L. and Warner P.P. 1990). If the standard assay could be modified for use with such a packing, its application to rapid off-line sample analysis would be greatly enhanced. Alternatively, should there be a need to improve protein recoveries, a polymeric stationary phase of lower hydrophobicity could be employed. One possibility would be to use a hydrophobic interaction chromatography polymeric stationary phase, such as TSKgel Phenyl-5PW (Toya Soda Co., Tokyo, Japan).

The mobile phase

The effect of changing the mobile phase pH and of including urea in the mobile phase buffers was described in section 3.2.2.2. Most other modifications to the mobile phase composition are unlikely to bring about a significant improvement in the standard assay; switching from acetonitrile to another solvent would alter solute elution times but would probably not improve resolution (Corran P.H. 1989, Salt D.E. et al., in preparation), using Guanidine-HCl in place of urea is anticipated to have no beneficial effect (Sharifi B.G. et al. 1985, Nugent K.D. et al. 1989), and lowering the mobile phase temperature would be expected to reduce resolution (Nugent K.D. et al. 1989). It is likely that protein recoveries could be improved by decreasing the mobile phase KH2PO4 concentration, since the phosphate acts as an ion-pairing agent, shielding the basic amino acid residues of proteins from the stationary phase (Guo D. et al. 1987). However, reductions in KH^PO^ concentration are likely to be limited by the loss of buffering capacity.

Gradient shape

While modification of the gradient shape was unable to bring about an improvement in resolution (section 3.2.2.3), it could be used to improve the standard assay by reducing the assay run length. This could be achieved by starting the gradient at a higher acetonitrile concentration (since few solutes were eluted below 15 % acetonitrile, this would represent a sensible lower limit), or by sloping the gradient more steeply over the regions where pl-381 elution does not occur. Starting the gradient at a higher acetonitrile concentration may have the additional benefits of increasing protein recoveries and removing the blank chromatogram peaks (Corran P.H. 1989). By eliminating the need to run blank gradients for the generation of difference chromatograms, removal of the blank chromatogram peaks would considerably reduce the time taken to analyze a large number of samples. 3,3.3.3 Purification of pl-381 by the standard assay

Because of its ability to rapidly perform high resolution protein separations, there is increasing interest in the use of RP-HPLC for large scale protein purification (Verzele M. et al. 1988, Verzele M. 1990). À scaled up version of the standard assay appears at first sight to be particularly attractive, since it has the additional advantage of separating pl-381 from interparticulate RNA (section 3.2.2.4). How e v e r , owing to the high cost of the stationary phase, an extensive program of research would be required on the existing analytical scale and on a semi­ preparative scale before scale up could be contemplated. Detailed information would be required on protein yields and purification factors, at analytical loadings (section 3.3.3.1), and at the much higher loadings commonly employed in preparative RP-HPLC (Verzele M. 1990), and if necessary optimization studies would need to be carried out. An assessment would be needed of covalent modifications to protein structure occurring under assay conditions (Volkin D.B. and Klibanov A.M. 1989), including eluent reinjection experiments. Last but not least, the major problem of reassembling the denatured pl-381 protein eluted by the

standard assay into native 5620 Ty-VLPs would need to be addressed.