and the protonated anion, and would therefore test this hypothesis of iron release. A change from Arg to Asp or Glu would introduce further possibilities. The negative charge is likely to prevent anion binding, but the carboxylate group could possibly substitute for the anion and bind directly to the metal ion. If this were the case there would be no possibility of protonation of the anion, and in fact no need for anion binding at all since the positive charge of Fe3+ could be partially
neutralised directly by the carboxylate group. Glycosylation of lactoferrin
Human lactoferrin contains three potential recognition sites (Asn -X-Ser{Thr) for the addition of N-linked carbohydrate groups. Of the three sites at Asn 137, Asn
478 and Asn 623 only the first two have been reported to be glycosylated in human tissues (Metz-Boutigue et al., 1984). The N-linked carbohydrate groups have been implicated in the indirect stabilisation of iron binding (Le grand et al.,
1990) and in receptor interactions. However, conclusions to the contrary have been drawn by other investigators (H.M. Baker pers. comm.; Padda and
Schryvers, 1990). Synthesis of non-glycosylated lactoferrin would allow the role of the carbohydrate groups to be examined under conditions where there was no possibility of other changes to the protein, such as can occur during enzymatic degl ycosy lation.
A further advantage of producing deglycosylated protein is that it may crystallise better than the native glycoprotein. This has already been shown in the case of intact human apolactoferrin (Norris et al., 1989). In this instance, the
carbohydrale groups were removed using the enzymes Endoglycosidase F (Endo F) and Peptide N:glycosidase F (PNGase) both isolated from Flavobacterium meningosepticum. The production of non-glycosylated protein in cells would simplify pmification as it would remove the necessity for enzymatic
deglycosylation. This involves several steps which always results in the loss of some protein and has the potential to affect the protein
in
unknown ways.Mutagenesis protocols
A wide variety of techniques are available for introducing site-specific mutations into cloned cDNA. Most of the earlier methods are reviewed by Leatherbarrow and Fersht (1980). Although variations of many of these methods are still used they have largely been superseded by the method described by Kunkel et al.
(1987) which Qses a dur, ung-strain of bacteria to produce the template. The advantage of this approach is that dur ung-strains produce DNA in which a high
A 4 . 2 A4.2 . 1
proportion of the thymine residues have been replaced by uracil. The lack of dUTPase (dut -) activity results in high intracellular levels of dUTP which
increases the frequency of uracil incorporation into the DNA. Once uracil has been incorporated into the DNA in these cells it is not efficiently removed because uracil N-glycosylase (ung-) is also inactivated. This enzyme is responsible for initiating the DNA repair mechanism which allows replacement of uracil by thymine in wild type cells. Single strand DNA which has been prepared from phage or phagemids grown in dur ung-bacterial hosts can be used as the template for mutagenesis by elongation from the annealed mutagenic oligonucleotide. Transformation of the reaction products, following elongation and ligation, into
dut+
ung+ bacteria will result in degradation of the uracil-containing template strand, ensuring that only the mutated strand survives.RESULTS
Production of a mutant
Cloning into p1Z and preparation of the template phage
Although pGEM:LfN2 (Section A2.2.4) would have been suitable for the preparation of single strand template phage, it was decided to clone the cDNA encoding LfN into pTZ1 8U, the vector supplied with the mutagenesis kit available. The main feature of pTZ1 8U is that, like pGEM-3Zf(+), it contains the f1 origin of replication, allowing production of phage-like particles containing ssDNA when cells have been superinfected with helper phage. The cDNA encoding LfN was excised from pGEM:LfN using EcoRl and BamHl and ligated into pTZ18U which had been cut with the same enzymes. After transformation (Section A1.2.6) into XL-1 cells a single colony was chosen, analysed by restriction enzyme digestion and then used to transform competent CJ236 cells which were
selected on plates containing ampicillin. This clone was called p1Z:LfN and a partial restriction map is shown in Fig. 36. Once a clone had been obtained in CJ236, uraeil-containing phagemids were prepared as described in section Al.3. 1 . Titration and extraction of the phage
Before the ssDNA was extracted from the phage they were titred in an attempt to estimate the level of uracil incmporation. Two bacterial strains, one dut+, ung+ and the other dur, ung-, were used to titre the phagemids. Approximately
HP
fold fewer colonies were obtained from MV 1 1 90 cells(dut+, ung+)
than from the CJ236 cells (dui, ung-). This difference was greater than the value suggested byFig. 36 Partial restriction map of pTZ:LfN.
Stop
the suppliers indicating that almost all of the phage contained uracil. If a large number of colonies are obtained in the
dut+, ung+
strain the template is not suitable for use in mutagenesis experiments as many of the parental strands will survive when transformed into thedut+, ung+
strain, decreasing the efficiency ofmutagenesis. The results obtained here suggested that ssDNA extracted (Section A1 .3.2) from these phage would serve as a good template for mutagenesis because
almost all of the parental strands would be degraded in a
dut+, ung+
host.Synthesis of the complementary strand
Oligonucleotides were obtained from commercial suppliers and if they had not already been purified they were purified as described in section A1 .2.2 1 . The oligonucleotides were then phosphorylated (Section Al.3.3) prior to addition ro the purified template at a ratio of between 20:1 and 10:1. The reaction mixture was then slowly cooled from 7rPC to room temperature (-1 hour). During this time the oligonucleotides annealed to the template. Once the primer had annealed the second strand was formed using the enzyme T4 DNA polymerase and the complete strand was ligated with T4 DNA ligase. T4 DNA polymerase was
chosen because it has no strand displacement activity preventing it from displacing the annealed oligonucleotide and cycling around on the same template strand (Masamume & Richardson, 1971). Other enzymes (Klenow and T7 DNA
polymerase) have higher processivity activities but they have the ability to displace the oligonucleotide resulting in a lower proportion of mutagenic clones (Biorad mutagenesis manual). Details of the annealing, extension and termination protocols are given in section A1.3.4. The sequences and position of all the mutagenic oligonucleotides used in this study are given in Appendix 4.