Impact of the expression system on the final structure and function of recombinant proteins

JOSÉ .V. CARRATALA, ; ANA ARÍS, ; JULIETA M. SANCHEZ; ELENA GARCIA-FRUITÓS; LAIA GIFRE-RENOM,; N.EUS FERRER-MIRALLES,

Boston

Encuentro; 15th Annual PEGS Boston; 2019

Cambrige Healthctech Institute

The production of recombinant soluble proteins with high stability and optimum specific activity is one of the main goals in protein engineering procedures. In this context, we have evaluated the influence of the bacterial expression system used for the recombinant expression on the final conformation, activity and structural protein features. Metalloproteinase-9 (MMP-9) has been used as a model protein and has been produced in two endotoxin-free expression systems: Clearcoli and Lactococcus lactis. Since MMP-9 is a prone-to-aggregate protein it has been purified using recent stablished protocols in our lab based on mild solubilization of Inclusion Bodies (IBs) followed by IMAC purification. Four forms of MMP-9 were obtained as4 different peaks during the IMAC purification from L. lactis, being the first peak the most active in DQ gelatin metalloproteinase assays. In contrast, ClearColi give rise to 3 protein forms distributed in 3 peaks after the chromatography but in this case the second peak was that one containing the most active protein in the DQ gelatin assay. If we compare the specific activity of L. Lactis MMP-9 with that of ClearColi, we observed that the MMP9 had 10 times more activity when produced in L.lactis than in Clearcoli. We have determined by Circular Dichroism that production conditions (in this case temperature) are a determining factor in relation to the physicochemical characteristics of the obtained protein. The spectrum at 30 ° C showed a profile compatible with the majority of conformations in alpha helix, more pronounced in the case of peak 1 than peak 2, However, the spectrum of the peak 1obtained at 37 ° C show a near minimum at 217 nm characteristic of suspensions in preferred beta sheet conformations. The soluble MMP-9 samples obtained from ClearColi and L. lactis were analyzed by Dynamic Light Scattering (DLS) to determine the protein size. As affinity increased in the nickel column (affinity), the size of the proteins present in each peak tended to increase. On the other hand, although peak 1 presents proteins of similar sizes independent of the strain from which it comes and the induction temperature, peak 2 shows greater dependence on the mentioned factors.The 4 peaks obtained in the affinity chromatography of MMP-9 produced in L. lactis were also subjected to a thermal stability test and the evolution of the fluorescence emitted by the Trp was determined. MMP-9 from peak 1 was the most stable since it has the highest unfolding temperature, this data coinciding with the peak that contains the most active MMP-9. On the other hand, peak 2 of MMP-9 from Clearcoli exhibit the lowest value of Trp fluorescence maximal wavelength expressed as CSM, suggesting the highest conformation quality and coincident with the highest active MMP-9 peak.In conclusion this study reveals that the final structural conformation and the consequent IMAC-purification profile and specific activity is totally influenced by the chosen expression system.