Cysteine-rich secretory proteins (CRISP) are monomeric proteins with masses of 20 to
30 kDa and 10–16 conserved cysteine residues. In this master's thesis, we aimed to
prepare a recombinant form of CRISP-1 from the venom of the nose-horned viper
(Vipera ammodytes ammodytes), designated VaaCRISP-1. We used the »SHuffle K-12«
strain of E. coli with an inserted pMAL-c5X plasmid. VaaCRISP-1 was expressed as a
fusion construct containing the maltose-binding protein (MBP5) at its N-terminal end,
followed by a cleavage site for activated factor X (FXa) and the sequence for
VaaCRISP-1 (UniProt ID A0A1I9KNM0_VIPAA) sans its signal sequence
(rVaaCRISP-1). After expression, the fusion protein was isolated using affinity
chromatography on an amylose resin column. Cleavage of the fusion product with FXa
was performed while it was still bound to the column or after elution. The intact
construct was stable, soluble, and clearly detectable on the gel after gel electrophoresis.
However, after cleavage with FXa, no product with a mass corresponding that of
rVaaCRISP-1 was visible on the gel. We assume this was due to the aggregation of
misfolded rVaaCRISP-1 molecules immediately after the removal of MBP5. Despite
the fact that E. coli SHuffle K-12 is specifically desgined for the production of proteins
containing multiple disulfide bridges, the folding of rVaaCRISP-1 was unsuccessful. By
purifying the sample on a C18 column using a HPLC system, we were nevertheless able
to isolate enough product to confirm its N-terminal sequence using Edman degradation,
confirming it as rVaaCRISP-1. Although we attempted to optimize the expression
conditions by adjusting IPTG concentration, temperature, and expression time, we were
unable to improve yields beyond 10 μg of product per liter of culture. Therefore, we
conclude that E. coli SHuffle K-12 is not suitable for the production of rVaaCRISP-1.
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