K Frase lexicalizada Sigla “Look at these nipples j.k you’re not seeing that today! Contexto: esta

The gene encoding Ssp DnaB mini-intein was successfully excised from the existing hybrid gene, leading to the generation of amySABDxhis. Restriction fragment analysis of plasmid isolated from transformed TOP10 E. coli indicated the presence of amySABDxhis (sans dnaB) as well as the functionality of the introduced BamHI restriction site, which was not previously present in the expression vector (Figure 26). DNA sequencing of amySABDxhis confirmed that there were no frame shifts or mutations, and that the dnaB sequence was no longer present in the hybrid gene, confirming successful excision of the intein encoding sequence.

However, transformed B. megaterium YYBm1 p1623amySABDxhis was unable to produce functional recombinant protein. Protein analysis by SDS-PAGE and anti-His-tag immunoblot was unable to detect AmyS:ABDx:His either intra- or extracellularly, and a starch degradation screen did not detect amylase activity in the extracellular protein of YYBm1 p1623amySABDxhis (Figure 28). Colony PCR confirmed the presence of amySABDxhis within transformed YYBm1, and DNA sequencing of the PCR product showed no frame shifts or mutations in the sequence of amySABDxhis that could explain why YYBm1 p1623amySABDxhis failed to produce the recombinant protein (Figure 27).

Further analysis of the recombinant protein sequence in collaboration with Prof. Geoffrey B. Jameson revealed a much greater problem with the protein. The structure of the C-terminal region of AlgX from A. vinelandii almost certainly involves the formation of a disulfide bond between the conserved C-terminal alginate-binding domain residue Cys466 and the conserved N-terminal acetyltransferase domain residue Cys348 by analogy to the disulfide bond observed for the equivalent residues Cys346 and Cys460 in P. aeruginosa (Figure 4) (Riley et al., 2013). However, Cys348 was not included in the recombinant protein design, as it was thought to lie outside the C-terminal alginate-binding domain (ABDx) of interest. Riley et al. (2013) considered that the disulfide bond linked the catalytic domain of P. aeruginosa AlgX to the carbohydrate binding domain. Thus in the design of the recombinant fusion protein, it was thought that the alginate binding domain did not require this disulfide link.

However, the results clearly indicate that this disulfide link is critical to the stability of folding of the alginate binding domain of the fusion protein. This left only two cysteine residues present in the final recombinant AmyS:DnaB:ABDx fusion protein: (1) the alginate-binding domain cysteine, Cys781; and (2) Cys564 located within the intein (Appendix I). Because of the additional 99 amino acid residues between the cysteines of the recombinant protein compared with AlgX, of which many vary from the original AlgX sequence, it cannot be expected that a disulfide bond would link Cys564 and Cys781. And if one did, the alginate- binding domain structure may not be folded correctly, and would be unlikely to function.

Disulfide bond formation is assumed to occur in the recombinant protein due to the aggregation of the protein during the DTT-sensitivity test (Figure 23). However, the disulfide bond that led to this aggregation is more likely to be the result of protein denaturation during preparation for SDS-PAGE when the protein sample is heated to 95˚C (Section 2.10.1.1). The denatured protein would be free to form new linkages that could link the cysteine residues at this stage.

Furthermore, this oversight may have caused the unsuccessful production of intein-free recombinant protein. With the removal of intein, Cys564 is also eliminated, and hence the ABDx residue Cys781 (Cys627 in AmyS:ABDx:His) has no other cysteine to link to. Without a disulfide bond, the ABDx domain would be incapable of folding correctly, and without aberrant intein activity the α-amylase domain would not be cleaved off. The improperly folded ABDx domain could then disrupt the structure of the AmyS domain, leading to the improper folding of the entire protein. Proteins that do not fold correctly typically undergo rapid breakdown in the cytosol for recycling of peptides. This would explain why His-tag was not detected in the immunoblot (Figure 28), as the polyhistidine motif was hydrolysed in addition to the misfolded protein.

However, given the high efficiency of the xylose promoter, low-level detection of His-tag would still be expected intracellularly as the recombinant protein would be produced so long as the inducer is still present, even if the protein is non-functional. It is possible that during molecular cloning, the p1623 vector was damaged, which caused significant inhibition of amySABDxhis expression. In particular, if xylA was damaged and sensitivity to xylose was lost, recombinant protein production would be drastically reduced. The occurrence of one of these factors, or a combination of both, would result in the failure to produce a functional

recombinant protein. It is recommended that the p3stop1623hp vector should be sequenced to examine sequence fidelity and integrity, and the amySABDxhis insert should be transferred to fresh p1623 plasmid, or a different vector entirely. Saliently, the gene for the recombinant protein should be redesigned to include the critical cysteine residue corresponding to P. aeruginosa AlgX Cys346, or to eliminate the naked cysteine (Cys781) of the recombinant protein alginate binding domain by, for example, converting the cysteine residue to a serine.