Sequencing o f expression vector (pG lD l) containing 33.H11 V h sequence.

DNA sequencing of the expression vector (pG lD l) containing 33.H11 Vh sequence was carried out using the Sanger dideoxynucleoside chain termination method (Sanger et al., 1977) using the T7 Sequenase (de-aza) kit, version 2.0 (Amersham), according to the manufacturers instructions. The primers used for sequencing were Hugl and Neo A.

H ugl 5 ’ TTGGAGGAGGGTGCCAG 3 ’

(binds to p G lD l 90bp 3’ of Vh region cassette)

Neo A 5 ’ CTCCATAGAAGACACCG 3 ’

(binds to p G lD l 30bp 5’ of Vh region cassette)

The reactions were run on a polyacrylamide gel (60ml acrylamideibis-acrylamide, 19:1 (w/v) gel solution (Amresco, Anachem, Luton, Bedfordshire, UK), 300pl 10% ammonium persulphate (w/v) and 30pl TEMED (N, N, N ’, N ’- tetramethylethylenediamine solution) (Sigma, Poole, UK) in a sequencing tank containing Tris Borate EDTA (TBE) buffer (45mM Tris-borate, ImM EDTA). The gel was exposed to X-ray film (Kodak, Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK) in a film cassette for 24 - 48 hours and developed using an automatic developer (Xograph Imaging System X4, Bedfordshire, UK).

2.5.2 Site-directed mutagenesis o f 33.H11 Vh expression vector using the QuikChange ™Site-directed Mutagenesis Kit (Stratagene, California, USA).

Through sequence analysis of 3 3 .H llV n/pG lD l, it was deduced that expression may be improved by reverting a proline (derived from an earlier PCR error) to a leucine by reverting a single nucleotide change from a C to a T in FR2 of the Vh sequence. This single nucleotide change was carried out using the QuikChange^^ Site-directed Mutagenesis Kit (Stratagene, California, USA) according to the manufacturers instructions.

The mutagenic primers used in this protocol were designed individually according to the desired mutation. The primers were made and purified by fast polynucleotide liquid chromatography (FPLC) by Genosys, Cambridge, UK. The primers used were:

R E IM FO R

5’ CAA COT CAA GAA CTC ACT GTA TCT GCA AAT GAA CAG CC 3’

REIM BA CK

5’ GGC TGT TCA TTT GCA GAT ACA GTG AGT TCT TGA CGT TG 3’

The dsDNA template used was the whole 3 3 .H 1 1 Vh/pG 1D 1 plasmid. The dsDNA was prepared using the QIAGEN QIAprep ® Miniprep kit (Crawley, West Sussex) as detailed in section 2.3.2.1. The DNA was linearised using a H indll restriction digest and the product was run on a 0.7% agarose gel in order to estimate the concentration of dsDNA in the preparation. As recommended by the manufacturers, a series o f sample reactions (5, 10, 20 and 50ng) using various concentrations were set up while keeping the amount of primer constant.

To confirm that the point mutation of a C to a T (proline to leucine) had been successful, DNA sequencing of the mutagenised expression vector was carried out as before (see section 2.5.1). The primer used was the 3’ primer JH4b FOR as shown below in Figure 2.5.

p G lD l 3 3 .H 1 1 Vh r e g i o n

3 3.H 11Vh/pG1D1 3 ' ÀÀC CTA GGT GAG TGG ACT CCT CTG CCA CTG GTC 5 '

P r im e r :

JH4b FOR 5 ' TIG GAT CCA CTC ACC TGA GGA GAC GG1 GAC CAG 3 '

Figure 2.5 Design of JH4b FOR for DNA sequencing of 33.H11 Vh

This primer binds to the last six codons of the 33.H11 Vh region and to the first five codons in

Materials and Methods

2,6 Transfer o f B3 Vh sequence from expression vector p G lD l to pG lD 210. 2.6,1 Background

The original heavy chain expression vector, p G lD l was used successfully by our research group and by its developers (AERES Biomedical, Mill Hill, London, UK) until the year 2000. At that point AERES Biomedical developed a new vector, pGlD210. Again due to commercial sensitivity, no published reference is available for this vector. This vector contained two potential improvements. The first was the presence o f the HCMVi enhancer 5’ to the insert, which had previously been shown to enhance expression (Chapman et a l, 1991).

Secondly, AERES Biomedical had discovered that the SA site in p G lD l was not 100% efficient. Using mass spectrometry it was shown that in some but not all proteins derived from COS-7 cells transfected with p G lD l, the intron between the SD site and the SA site was not spliced out and consequently some heavy chains contained an extra 22 amino acid region between Vh and Ch. This extra 22 amino acid region was only detected because this sequence encodes a glycosylation site. (Normally a difference of 22 amino acids would not be detected by mass spectrometry). In the improved vector, pGlD210, the SA and SD sites were removed such that the Ch sequence followed directly after the Vh (see Figure 2.6 for pGlD210 vector map). Although neither our research group nor AERES had noted any functional problems associated with the presence of this intron, we decided that ideally our Vh sequences should be transferred into the new pGlD210 vector to take advantage of these potential improvements. All of the expression experiments in this thesis were carried out with the original p G lD l vector. However for future reference the whole IgG expression levels and anti-DNA binding activity of BSVn/pGlDl and B 3 Vh/pG 1 D 2 1 0 are compared in this thesis,

firstly to identify whether the HCMVi enhancer did improve expression and secondly to ensure that the vector used did not affect IgG binding activity.

Figure 2.6 Vector map of recombinant heavy chain expression vector, BSVn/pGlDZlO

pGlD210 contains the human cytomegalovirus (HCMV) promoter to drive transcription of the recombinant immunoglobulin gene, the SV40 origin of replication to give high levels of transient expression in COS-7 cells and the mouse dhfr gene coding sequence driven by the SV40 early promoter to act as a dominant selectable marker during stable transformation. The SV40 promoter that drives the dhfr is crippled by the presence of a defective SV40 promoter- enhancer sequence so that expression is poor thus allowing for the selection of high expression level clones using comparatively low levels of methotrexate. pGlD210 also contains an ampicillin resistance gene (AmpR) driven by an internal promoter to enable it to be cultured in E.coli and a cloned PCR fragment that encodes the human yl constant region (Cyl). The Vh

region sequence of B3 was cloned into expression vector pGlDl immediately 5’ to the Cyl

Materials and Methods N hel 7 6 4 5 EcoRI 1 B glll 6 7 9 0 Mlul 1529 BstElI 1534 H indlll 1535 dhfr N h el 6 2 9 0

HCMVi prom oter

Human Heavy Chain Variable Region SV40 early prom oter

B 3V H /pG lD 210 SV40 Origin 7651 bp Xhol Human yl constant region Am pR

AmpR prom oter

N hel 3 2 9 0 Pvul 3 6 9 0 KEY SA SD AmpR dhfr HCMV Promoter Gene

Human heavy chain variable region Human y 1 constant region DNA sequence Immunoglobulin leader sequence

Splice acceptor site Splice donor site

Ampicillin resistance gene Dihydrofolate reductase gene Human cytomegalovirus

Most restriction sites are shown in black. Those written in red indicate that they were used for cloning described in this thesis.

2.6.2 Production o f a X h o l site in p G lD l/B 3 Vh by PCR.

The expression vector pGlD210 has a multiple cloning site that contains the restriction sites, H indlll and Xhol. As there are no SA and SD sites in pGlD210, to transfer a fragment that contained the Ig leader sequence and the B 3 Vh region sequence into pGlD210, a X hol site was introduced into the end o f the B 3 Vh sequence without changing the amino acid sequence by PCR, as described below. The primers were made and purified by fast polynucleotide liquid chromatography (FPLC) by Genosys, Cambridge, UK. The primers used were:

CIH BACK 5’ GAG CTA AGC TTG CCG CCA CCA 3’ IBH FO R 5’ CTC ACC GCT CGA GAC GAT GAC CAG 3’

The primer CIHBACK binds to the sequence o f p G lD l 5’ to the Hindlll site (i.e. outside the leader region whilst the primer IBHFOR anneals to the last six codons o f B3 Vh insert in p G lD l, as shown in Figure 2.7.

B 3 Vh L V T V S S

pGlDl