Normas Generales

In this thesis, the production of the previously designed and engineered variants of ProCA1 from Dr. Anna W. Maniccia and Dr. Jingjuan Qiao’ s dissertation, and ProCA32 variant from Dr. Shenghui Xue’s dissertation are discussed in detail in Chapters 2-5. Specifically, the objective of Chapters 2 and 3 is to seek optimal conditions through: change in parameters during expression and purification methods for obtaining high yield, low toxicity, high purity, sTable bioactivity of protein of interest through an effective, low cost procedure. This applies as well to all other ProCAs that will be mentioned throughout this study. In Table 1.3, each charged variant tackled in this study is summarized with its associated mutation, purpose for the mutation, and related Chapters.

In Chapter 2 of this thesis, expression conditions for tag-less ProCA1 charged variants are analyzed by change in host cell strain. Competent cell strains used are BL21 (DE3), BL21 (DE3) pLysS, Tuner, and Rosetta-gami (DE3) pLysS. Henceforth, these cell strains are named BL21, BL21 (DE3) pLysS, Tuner, and Rosetta-gami pLysS throughout Chapter 2 and 3. The tag-less expression method demonstrates low molecular weight at ~ 11.2 KDa for each ProCA1 charged variants, which are listed below in Table 1.2. Chapter 2 describes the effect of recombinant protein production is dependent on the used host cell strain. Each cell strain has a deletion mutation, which gives them a specified characteristic during expression system. For instance, ProCA1 charged variants expressed in BL21 (DE3) pLysS cell strain demonstrate lower production at higher molecular weight compared to BL21 and Tuner. Tuner is dependent upon the concentration of inducer added during the expression and this phenomenon is demonstrated from the expression gels and final yield post FPLC-Q column isolation (Section 2.3 of Chapter 2). Further isolation of the concentrated pure samples is passed through a size exclusion high-pressure column but the yield is decreased dramatically.

The expression and purification conditions of GST-tag charged ProCA1 charged variants and targeted ProCA1.affibody variants are tackled in Chapter 3 of this study. The following parameters for

expression system are analyzed: a) the temperature post induction, b) the inducer (IPTG) concentration, and c) the host E.coli cell strain. The following parameters for the GST-tag purification system are analyzed: a) 100 mg lysozyme/per 1 L culture, b) use larger diameter column, c) use 10 mM DTT and 0.05 % triton X-100, and d) use of a refolding method on inclusion body GST-tag ProCA1 charged variants and ProCA1.affibody variants. Summarized expression and purification data for GST-tag ProCA1 charged variants through change in conditions/parameters are listed under Section 3.3 of Chapter 3. The yield from each host cell strain and purification conditions for all variants charged and targeted is summarized in Section 3.3.

In addition to obtaining an optimal condition for expression and purification, the structural and functional characteristics are summarized in Chapter 4. The secondary and tertiary structural conformation stability of the ProCAs is tested through monitoring the secondary structure with circular dichroism and tertiary hydrophobic folding with tryptophan-fluorescence emission spectroscopy. The functional characteristics involve determining the metal-binding affinity of the binding pocket to the lanthanide metals Gd3+ and Tb3+. The metal binding study for Tb3+ is observed through the following two

fluorescence methods: a) a buffer system titration method, and b) a competition assay using FLuo-5N dye. The summarized Kd for each of the titration methods for ProCA1 variants targeted and charged are compared in a Table that is available in Section 4.3 of Chapter 4. The competition method is applied for Gd3+ binding affinity determination with the use of a Fluo-5N Invitrogen commercial dye. Furthermore,

the relaxation properties are measured with change in ProCA1 variants to Gd3+ concentration ratio, and

analysis of relaxation rate change in relation to ProCA1 variants concentration change with the fixed Gd3+ concentration. The ProCA32 variant has a different scaffold protein and two binding pockets

compared to ProCA1 variants mentioned previously.

The conventional bench work purification system of the fed batch fermentation for ProCA32 is analyzed and compared to two other systems. Specifically, the conventionally expressed ProCA32 and

fed-batch fermented ProCA32 pellets are compared through the typical bench work purification system. In addition, the ProCA32 pellet from fed-batch fermentation is purified through the use of a specified PEG concentration. Essentially, all three tactics for purification are compared in terms of overall yield after FPLC-Q column isolation. The relaxation properties at varying ratio of the ProCA32 to Gd3+

concentration are also compared for each sample obtained from the individual expression/purification systems. The purified ProCA32 from fed-batch fermentation is analyzed for structural and functional stability through an NTA-buffer system titration and a competition assay with FLuo5N titration using Fluorescence spectroscopy. Chapter 6 describes the goals achieved; the challenges, and the major findings from this study overall.

Table 1.3 The summary of ProCA variants used in this study [20, 29, 34-36].

All ProCAs of the rat scaffold protein CD2.D1. All ProCA1 charged variants are referred as: ProCA1, 7E15E, 7E15N, and 7E15Q for the design approach in the binding pocket. The grafting approach at the c-terminus of ProCA1 is represented based on ProCA1.affi342 targeted towards HER2/neu and ProCA1.affi1097 targeted towards EGFR cancer biomarkers. The derived ProCA32 represents the 3rd generation of designed protein based contrast agents and holds two binding pocket compared to the first generation ProCA1 that has one binding pocket design on its scaffold’s surface.

2. EXPRESSION AND REFOLDING PURIFICATION OF TAG-LESS PROCA1 VARIANTS IN VARIOUS E.