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4.2. Affimer Protein Expression and Purification
As described in section 3.2.2, for initial screening purposes Affimers were produced in 50 mL of
autoinduction media and purified by nickel affinity chromatography. This provided a sufficient
protein yield to perform nitrocefin assays in triplicate on a single plate. Once selected and subcloned
into pET11a vectors, Affimers were produced in 500 mL culture volumes and purified by nickel
affinity chromatography. This yielded greater protein expression, commonly greater than 10 mg of
protein per culture which was enough for several assays in both biological and technical triplicate
repeats. One advantage of Affimers is their high yield from E. coli cells, which is advantageous over
other technologies such as the production of antibodies and was evident in this study Coupled with these findings, the nickel affinity chromatography as a purification method appeared
to be efficacious, yielding high amounts of protein in pure samples. Bands on SDS-PAGE gels were

clear signifying their expression, with little - to no - protein contamination.
4.3. Affimers as inhibitors of Metallo-ß-lactamases
In this thesis, inhibition of the rate of nitrocefin hydrolysis by VIM-1 was accomplished with
Affimer61, reducing the rate by 47%, which suggests that Affimer reagents can modulate the activity
of VIM-1. Further characterisation must be performed on this and other Affimers, to establish an IC50
and determine the method of inhibition. An IC50 denotes in this case the amount of Affimer required
to inhibit the hydrolysis of nitrocefin by VIM-1 by 50%. This would be established by performing a
series of nitrocefin assays utilising a constant VIM-1 concentration, with an initially high (in order to
establish maximal inhibition, close to 100%) but decreasing concentration of Affimer reagent.
Technical and biological repeats would be necessary as within this study, to establish at what
concentration of Affimer is VIM-1 inhibited by 50%. The lower this value, the better the Affimer
would be since less of the reagent would be required to reach this inhibitory level.
Structural characterisation of the Affimer-target complex would shed light on the binding
interactions and the method of inhibition of Affimer 61. Identification of the binding site of
Affimer61 could also be referenced against known sequences of VIM-variants to establish the
likelihood of whether it could be used as an inhibitor against these also.
The use of alanine Affimers in this project is consistent with other controls used in Affimer projects.
The truncated variable regions, consisting primarily of alanine to allow structure and flexibility
without complex sidechain interactions, allows for establishing during assays that the basic structure
of the Affimer itself is not responsible for perceived effects. In the case of this project, it was to
ensure that the Affimer structure was not able to either hy

Discussion
As the global spread of antimicrobial resistance continues to increase, so too does the need for
novel reagents that are able to detect, bind, and modulate resistance mechanisms. Inhibitors that
target metallo-ß-lactamases are highly sought-after as their function allows them to threaten the
efficacy of our current clinical arsenal of antibiotics.
Affimer reagents have previously been found to inhibit the activity of NDM-1, a metallo-ß-lactamase
(MBL) with a functional similarity to VIM-1. In prior studies at the University of Leeds inhibition of
NDM-1 was found to be up to 85% at five-fold the Affimer. A. Herbert and Dr L. Faveri identified
Affimer 21 which inhibits NDM-1 by a non-competitive mechanism[88][87]. This work investigates the
proposal that Affimer reagents could be found to have a similar inhibitory effect on VIM-1.
4.1. VIM-1 Protein Expression and Purification
Initially VIM-1 proteins were expressed in BL21 Star (DE3) cells, which yielded a good quantity of
protein, around 3 mg per 500 mL culture. Initially VIM-1 was chemically biotinylated through the use
of EZ-link NHS-SS-Biotin, which binds primarily to lysine residues, of which there are six in VIM-1.
Chemical biotinylation is a common method of biotinylating proteins prior to immobilisation on
streptavidin coated surfaces in preparation for phage panning in phage display and phage ELISA[81]
.
In this study chemical biotinylation was found not to be successful (see Results 3.1.2.) An alternate
method of protein biotinylation is to add a biotin acceptor peptide (BAP) tag to the terminus of a
protein sequence. In strains containing the BirA plasmid, an expression plasmid for BirA ligase, the
BAP tag is biotinylated. For this reason, AVB101 cells were used for the expression of biotinylated
protein. Expression in AVB101 cells showed a protein yield of 2.8 mg per 500mL culture. The extent
of biotinylation of the resulting protein was analysed via Western blot and ELISA assays.
It should be noted for future work on preparing biotinylated protein for the purpose of protein
immobilisation, is that the genetic BAP-tag approach results in a lower diversity of orientations of
the immobilised protein compared to that of chemical biotinylation. As such, it may be that though
the results of the phage display were better than when using chemical biotinylation (such as through
NHS-SS-Biotin) it may represent in other cases a reduced ability to find a suitable Affimer or other
binder that would instead have bound to a -now secluded- section of the protein. This should also be
a consideration when proteins such as metallo-ß-lactamases are being used, as covering the active
site of an enzyme could reduce the potential of finding a broad-spectrum Affimer reagent that acts
as an inhibitor

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