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pvdQ Protein Expression

 

To express the pvdQ protein, it was cloned into the pET21a (+) expression vector. The Pet21a (+) was digested with NotI to yield a linearized 5443bp fragment. Dephosphorylation of the vector fragment using calf intestinal alkaline phosphatase was carried out to prevent relegation. Then, the pvdQ-B0015 gene in the pSB1AK3 vector was also digested using NotI, yielding the desired fragment size of 2,400 bp.

 

 

 

The two digested fragments were then ligated. Since both fragments yielded NotI sticky ends, performing restriction digestion to check the orientation is essential. The ligated product of correct orientation when digested with ScaI and DraIII would yield fragment sizes of 660, 2014, and 5237 bp. When in correct orientation, digestion with SalI would yield bands that are 952 and 6959 bp. The fragments ligated in an incorrect orientation would yield fragments of 660, 1277, and 5974 bp when digested with ScaI and DraIII. When digested with SalI, it would yield fragments of 1542 and 6369 bp.

From the gel photo, it can be deduced that Sample 3 yields the desired band sizes, and therefore produces the correct ligation product.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The pvdQ protein is subject to posttranslational processing resulting in its autocatalytic cleavage into separate alpha and beta subunits. The pvdQ sequence used in this project consists of a His tag sequence at its C-terminal. Thus, after cleavage, only the beta chain can be detected by Western blotting techniques using an Anti-His tag primary antibody derived from mouse. The untagged alpha subunit will not be detected.
 

From the developed X-ray film, it can be seen that the 60kDa band denoting the beta chain is missing from Lane 3, which is the IPTG induced BL21 whole cell lacking the recombinant pet21a plasmid.  Lanes 4 and 5 are the supernatant and pellet respectively after sonication of the IPTG induced BL21 containing the plasmid. The presence of the 60kDa band indicates that half the protein exists in its soluble form and the other half is insoluble. The pvdQ sequence consists of a signal peptide sequence that translocates the protein to the periplsmic space of a cell. After performing osmotic shock, the supernatant makes up the cell’s periplasmic fraction. Since the beta chain is detectable in Lane 6, it can be deduced that pvdQ is indeed translocated. Thereby, from this western blot, we were able to successfully clone pvdQ into the Pet21a vector and induce its expression.

 

 

The polyhistidine tag on pvdQ can be used for affinity purification. After inducing expression of the protein and subsequent sonication to lyse the bacterial cell, the pellet can be re-dissolved in an appropriate volume of elusion buffer. The lysate contains the soluble recombinant protein as well as other proteins from the bacterial host. It can be applied to a Ni-NTA His-Bind Resin that is made up of bound nickel ions to which the His-tagged protein binds with micromolar affinity. The proteins that are unable to bind with the resin are removed by washing with phosphate buffer with a 20mM of imidazole. Then, the His tag proteins can be eluted using a higher imidazole concentration ranging between 150-300mM. The bands in Lanes 3 and 11 are slightly larger than the 50kDa standard in Lane 1. It can be seen that the 60kDa beta chain is present in the sonication supernatant and in the purified elusion. All the His tag protein efficiently bound to the column resin because there isn’t any trace of protein in Lanes 5 to 10.