Análisis in silico del promotor de ipdC de A brasilense Sp7

As was described in the introduction, MRP- 8 and M RP-14 are very highly expressed

proteins which only recently have started to reveal their function. Some years ago, murine M RP-8, also known as C P-10, was found to be a highly potent chemoattractant

(137) for neutrophils. Human M RP- 8 is not chemotactic (138).

Functional data about murine M RP-14 is sparse although M R P-14 is reported to exhibit MDF-like activity (1) and, when coupled to beads, to recruit neutrophils over a 2 week period, in vivo (281). Previous work in this laboratory has shown that human M RP-14 is a novel activator o f M ac-1 and moreover, that human M RP- 8 negatively

regulates this activation by the formation o f a MRP-8/MRP-14 heterodimeric complex (198).

The aims o f this thesis are to further investigate the function o f M RP- 8 and

M RP-14 by the cloning, purification and biological characterisation o f the recombinant proteins and also by the generation o f knockout mice (described in chapters 5 and 6).

The rationale for cloning the proteins must first be addressed. It was decided to clone the murine proteins, even though the laboratory had both human MRP- 8 and

M R P-14 recombinant proteins and also some human functional data. This decision was taken, firstly, to allow comparison o f biological function between the human and murine proteins so that the relevance o f any conclusions drawn from the knockout models could be evaluated. Secondly, production o f the murine proteins was undertaken to allow the generation o f both polyclonal and monoclonal antibodies which would potentially be very important for both recombinant protein experiments and the future characterisation o f the knockout mice.

This chapter aims to describe the cloning and purification o f murine MRP- 8 and

M R P-14 and also to define some o f the basic biochemical features o f M RP-14.

3.2 cDNA cloning o f MRP-8 and MRP-14

Initial attempts to clone MRP- 8 and M RP-14 cDNAs centred around the use o f 5’- and

3 ’- primers which both contained a Ndel site, situated 5 ’- and 3 ’- to the regions of coding homology, respectively. Using this Ndel site, subsequent PCR products could then be cloned into the protein expression vector pET-3a. This vector is a simple,

Chapter 3:- Cloning and purification o f recombinant M RP- 8 and M RP-14

early-generation, protein expression vector which contains only a C-terminal T7 tag, which is not utilised if cloning a sequence with Ndel termini and a stop codon. The cDNA termini o f M RP- 8 and M RP-14 do not naturally contain N del sites, so

homologous sequence was supplied either side o f the engineered site to allow priming. A schematic o f the primer structure is shown in Figure 3.1 (on page 110).

PCR products were generated using these primers and a bone marrow cDNA library. The products were then genecleaned and digested with Ndel, to allow cloning into the Ndel-linearised pET-3a vector. The results o f this strategy were disappointing. Few products were cloned and those that were cloned, upon initial analysis were the correct length for the gene in question, 270bp for M RP- 8 and 368bp for MRP 14.

However, after sequencing, these clones were shown to be genetic artefacts, that is to say they did not contain MRP-8, M RP-14 or any recognisable DNA sequences, just

apparently random bases with assorted stop codons.

The reasons for the initial failure to clone the cDNAs may be threefold. First, as a general rule, enzymes require 6 bases pairs on either side o f their recognition site to

cleave efficiently (NEB, technical information) and only 4 were provided on the 5’- (for the forward) and 3 ’- (for the back) in the original primer sets. Therefore, o f the

products that were produced, many may have been lost at the cloning step, as they may never have ended up with termini which could be ligated. This would reduce the number o f clones available for analysis. Secondly, the failure may have been due to the short length o f the primers, internal to the cDNA sequences (18 bases) or to a failure o f the 5’- most or 3 ’- most regions o f the primers to anneal to template because o f the restriction site insertion. This would increase the chance o f cloning non-specific products. Thirdly, the failure may have rested with the template. Many cDNA libraries do not contain cDNAs o f less than 500bp in order to minimise the cloning o f cDNA artefacts within their system. However, consultation with the makers o f the library (D.Simmons, Oxford) suggested that small cDNAs should indeed be represented. Furthermore, probing o f the library with those primers used to generate specific genomic probes for M RP- 8 and M RP-14 (8F1/8B2 and 14F2/14B2, see section 5.2.1, page 192

and section 5.3.1, page 208) suggested that the M RP- 8 and M R P-14 cDNAs were

present within the library (data not shown). Taken together, these data suggested that a small change in approach was necessary to clone the M RP- 8 and M RP-14 cDNAs.

Chapter 3:- Cloning and purification o f recombinant MRP-8 and M RP-14 The second approach used, to clone the M RP- 8 and M R P-14 cDNAs, utilised

the pAMP-1 vector, specialised for the cloning o f PCR products. Use o f this vector necessitated using primers with specialised ends (described in 2.14.2.4.1, page 82) A schematic o f the primer structure is shown in Figure 3.2 (on page 110). Other primer

modifications included the addition o f an extra internal codon for both the forward and back primers (to increase specificity) and the relocation o f the added restriction site so that it did not interrupt regions o f complementarity and hence potentially interrupt annealing. In addition, a 3 ’- BamHI site was introduced so that cloning could be

directional, (5’- Ndel, 3’- BamHI) which avoids a second screening step for sense strand orientation. New template DNA was also used, in tandem with the bone marrow cDNA library; EST clones o f MRP- 8 and MRP-14 (172) were identified from a web database

search and sourced from IMAGE corporation, USA.

PCR products generated from the new template and also from the bone marrow cDNA library were genecleaned, cloned directly into pAMP-1 and transformed into E.coli XL-1 Blue. Colonies were then screened with the internal primers 8F1 and 8F2 for M RP- 8 and 14F2 and 14B2 for MRP-14. Positive colonies were then sequenced

and positively identified (data not shown). M R P-14 and a mutant M RP- 8 were cloned

from the bone marrow cDNA library (MRP- 8 had amino acid 79 mutated from F to Y,

known as M RP-8FY), M RP- 8 was cloned from the M RP- 8 EST clone. M RP-8, MRP- 8FY and M RP-14 cDNA clones were subsequently transferred into the pET-3a vector,

for improved protein expression, by virtue o f the 5 ’- Ndel and 3 ’- BamHI restriction sites and again transformed into E.coli XL 1-Blue. These clones were known as 3a-8, 3a-

8FY and 3a-14.

Colonies were again screened by PCR and positive clones re-sequenced (data not shown). Final positive verification o f identity was followed by transformation into E.coli. BL21 (DE3) pLysS, which is favoured for protein production rather than cloning.

The choice o f protein expression bacteria was based on the presence o f the DE3 plasmid in BL21 which allows IPTG induction o f the T7 polymerase and also the pLysS plasmid which prevents leaky expression o f T7 polymerase, and hence protects the bacteria from potentially harmful protein products.

The cDNA sequences for M RP-8, M RP-8FY and M RP-14 were later

transferred into the His-tag vector, pET-28a. This was achieved simply; sequences were excised from pET-3a by Ndel/BamHI excision, and cloned into pET-28a which had

Chapter 3:- Cloning and purification o f recombinant M RP- 8 and M RP-14

been linearised with Ndel/BamHL Vectors were then transformed into XL-1 blue. Recombinant colonies were assessed by PCR analysis with internal primers, as described above and positive recombinants were then transformed into E.coli. BL21 (DE3) pLysS. These clones were known as 28a-8, 28a-FY and 28a-14.

A.