The prokaryotic expression o f hCGh was investigated to determine a suitable system for the analysis o f mutant hCGB molecules. First a mutagenesis method had to be estabhshed to generate molecules which may be analysed for the loss o f B-cell epitopes. At the start o f this investigation the three-dimensional structure of hCGiJ was not known. Random mutagenesis was therefore employed to analyse residues in^ortant in epitope formation. Random mutagenesis using degenerate PCR primers has been used previously to
successfully identify epitope-loss mutants o f CD2 (Peterson et al., 1987). Random
mutagenesis has also been achieved by UV hght (Drobetsky et al., 1993; Poirier et a i,
1993), DNA polymerases which lack proof-reading activity (Liao et al., 1990), PCR with
nucleotide derivatives (Ueda et al., 1995) or chemicals such as N-methyl-N’-nitro-N-
nitrosoguanidine (Potvin et al., 1991), hydroxylamine (Kang et al., 1990) or
methoxylamine (Kadonaga et al., 1985). In this investigation methoxylamine was used
and its suitabihty for random mutagenesis o f hCGB assessed.
The analysis o f mutant hCGli molecules has certain requirements. The hCGh must be expressed in a system that ahows, 1) correct folding of the native molecule, 2) ease of production and expression o f mutants, 3) simple and rapid screening o f mutants with a panel o f mAbs. Prokaryotic expression o f foreign protein has been used for over 20 years
(Cohen et al., 1973) and the use o f E.coli as a host species is weU estabhshed The
mechanisms for controlling gene expression in this weU characterised organism are reasonably understood. There are many laboratory strains which are known to stably maintain plasmids. Several systems are commercially available, catering for a variety of
expression requirements and E.coli does tend to very efficiently over-express cloned
genes. E.coli also has a short doubling time and thus experiments can be done relatively
quickly. The different types o f expression systems for foreign proteins cater for different needs. Some produce a chimeric recombinant protein with the desired protein fragment fused with subunits o f other proteins. The resulting fusion proteins have particular
gene with the mutant protein. Two différent prokaryotic expression systems were explored here, pMAL and phage display.
In the pMAL system the cloned gene is inserted downstream o f the malE gene in a
translational reading frame. The malE gene encodes maltose binding protein (MBP).
When expression is induced from the efficient Puc promoter, a fusion protein results consisting o f the cloned gene product with MBP at its amino terminus (Maina, 1988).
This allows the recombinant protein to be purified from E.coli proteins using a maltose
column. There is a cleavage site for Factor Xa between the MBP gene and the cloned gene so that the MBP can in principle be removed from the protein o f interest (see figure 3.4).
By using different vectors, the fusion protein may be directed to the bacterial periplasm or be retained in the cytoplasm The pMALp vector has the hill length signal sequence o f the
malE gene which directs the fusion protein to the periplasm In pMALcRI this signal
sequence is deleted so that fusion proteins are retained in the cytoplasm Exporting the fusion protein to the periplasm may be advantageous for proteins which require the formation of disulphide bridges for correct folding, as these may not be formed in the reducing environment of the cytoplasm. hCGp, which has six disulphide bonds, may be one such molecule.
The M13 hlamentous bacteriophage system was also examined. This system was first used in 1985 (Smith, 1985). The protein o f interest is cloned into a phagemid between the phage pelB leader sequence and the phage gene m . Gene HI encodes a phage coat
protein glUp, present in three to five copies at the tip o f the bacteriophage (Goldsmith et
al., 1977). The glHp/cloned gene fusion protein is directed to the periplasm by the pelB
leader sequence. Upon superinfection with helper phage, the fusion protein is assembled into the phage coat along with the native gHIp encoded by the helper phage and a single stranded copy o f the phagemid may be packaged into some o f the phage particles (Figure 3.1). The secreted phage have on their surface a mixture o f normal ginp and ginp fusion protein. The bacteriophage can then be enriched for phage expressing the fusion protein by panning against specific antibodies. This system could be adapted for screening
Male E.coli harbouring phagemid
pH EN system
/
growth to log phasepFAB system
glucose repression removed
helper phage added IPTG added - expression
of fusion protein
fusion and phage proteins exported to periplasm, ssDNA replication occurs, assembly of phage particles with fusion protein, and secretion of recombinant phage
helper phage superinfect, ssDNA replication occurs, assembly of phage particles with fusion protein and secretion of recombinant phage
Figure 3.1 Two phage expression systems.
After growth o f E.coli culture containing the phagemid to log phase, there are slight differences in the two systems. In the pHEN system the glucose repression is removed and helper phage are added. These superinfect the bacteria via the sex pilus and ssDNA replication is initiated. The glllp fusion protein is produced and transported to the periplasm where it is incorporated with native phage proteins encoded on the helper phage. Recombinant and wild type phage are assembled and secreted. The pFAB system differs only in that IPTG is added to induce glllP fusion protein production before helper phage are added.
mutants to isolate those which had lost an epitope. If a mixture o f randomly mutated DNA is sub-cloned into the phagemid, the mutated protein will be expressed on the surface o f the bacteriophage. By panning the bacteriophage with mAbs it wiU be possible to select those which express hCGli. Those which have lost an epitope and hence no longer bind a mAb may then be selected from this population. This has the potential to be a very powerfrd screening system since bacteriophage are generally found at
concentrations o f 1 x 10^ V ml. It also has the advantage that when selecting the mutant protein, the DNA encoding it is also obtained.
Two bacteriophage expression plasmids were examined, pHENl and pFAB3. pHENl
(Hoogenboom et al., 1991) has an amber stop codon before gene HI, giving two possible
products. A truncated fusion product is produced excluding glUp if grown in a non-
supressor E.coli strain such as HB2151 which supports the amber codon. A frill length
frision protein is produced if expressed in an E.coli strain having the phenotype supE, such
as TGI, which suppresses the amber stop codon. The HB2151 strain, which lacks the amber suppressor, is used for making soluble product. pFAB3 is an alternative phagemid. It contains a truncated form of gene m , where the amino-tennmus is deleted. It has been proposed that large fusion partners exceeding 110 amino acids may lead to excessive
breakdown o f the fusion protein or impair glUp function (Parmley et al., 1988). Using a
truncated gene m may therefore facihtate expression of larger inserts. Another reason for deleting the N-terminus is that this is the part o f gHIp which renders the bacteria resistant
to phage infection (Crissman et al., 1984).
The various expression systems were examined in order to assess their suitability for screening large populations o f randomly mutated hCGfr clones with the aim of identifying epitope-loss mutants.