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Laboratory 7 Protein Purification of a His-tagged Protein,

Laboratory7: Protein Purification of aHis-tagged Protein,

ProteinDenaturation Studies, GFP sequence Analysis

Weused Ehwa, and Kevin’s data for GFP sequence Analysis since we didnot observe any significant mutations in our sequence. We assumedthat such errors occurred because mutations had taken place in thepromoter sequence instead of nucleotides in the main body of the GFPgene. Since the promoter was not investigated by BLAST Global Alignfunction, we obtained nearly 99% identical DNA and protein sequencefrom the normal sequence.

  1. We used an enzyme called lysozyme to break down the bacterial cell wall. A gentle method to break open the cells was necessary in order to keep the proteins folded. The cells had to be broken carefully to avoid damage of the cell contents. If we had just heated the cells, we could have broken open the cell wall but the proteins would have turned into a disrupted form instead of its native form which we aimed to purify.

  2. We centrifuged the liquid culture of bacteria cells the pallet was florescent. The first centrifugation separated the bacteria from the liquid that it was grown in. The pallet was bacteria and the liquid was supernatant. Since GFP was not secreted protein but rather kept in cytoplasm of bacteria cell, only the pallet glowed.

  3. We treated the sample with lysozyme and centrifuged it to obtain fluorescent supernatant. This is because lysozyme broke open the cell membrane of the bacteria leading to GFP protein being secreted out of the cell. Thus, the supernatant containing GFP glowed green. The pallet was just empty cell membrane of the bacteria cell.

  4. My group performed (b) and (d): (b) to raise the temperature of the protein to 80 °C, and (d) to change the pH of the solution by adding Acetic Acid. We took around 8 minute to fully denature the protein for part (b). Other two groups that carried out part (b) took 5minute 30 seconds and 3minutres, both of which took shorter time to denature the protein than our group did. Such difference is very significant since even 30 seconds difference is considered long when carrying out protein denaturation experiment. Next, we used around 50 microliters to denature the protein with Acetic Acid. Other groups who used the same procedure also obtained similar results ranged from 40 microliters to 70 microliters. We were in between the minimum and the maximum amounts compared to the results obtained by other groups this makes our results to appear acceptable.

  5. The most likely error to occur would have been the measurement error. Some groups might have stopped adding acid or the heating process while it was still glowing. The difference in measurement would hence increase because they did not make a correct judgement on whether or not the solution had lost fluorescence. In addition, some groups might not have mixed their content very well or pipetted different volumes of protein, which could affect the data and hence the results obtained.


Theoriginal colony that we picked was white. From the experiment weconducted, according to the DNA results, we found that there were 8nucleotides which were not identical with those of normal sequenceand subject sequence. However, we were informed that 30-40 bases fromthe ends of the sequence are usually not very good, as mentioned onpage 64 of the pre-lab handout. In addition, we were told toinvestigate only up to 800 bases. After excluding the irrelevantnucleotide changes, we came up with a conclusion that my group’ssequence had a total of three mutated bases at 168, 657 and 729 basepositions on the subject sequence. Because the original DNA stranddid not show any nucleotide at 729 base, we were made to believe thatthere might have occurred a programming error. The mutations atposition 168 and 657 are both substitutions, turning from C to T at168 and from A to G at 657. Such mutations occurred because we usedan error-prone polymerase. The header information provided that thepercentage identity between the two sequences turned out to be 89%,meaning that 89% of the bases are matching. Next, according to theamino acid result, our sequences had two mutated amino acids, whichwere located at 54thand 217thpositions of the amino acid sequence. 54thamino acid of the normal sequence is Proline but we had Leucine atthe position instead. At position 217, instead of having Histidine,we had Arginine.


Having a white colony was an indicationthat our GFP was not fully functioning as GFP protein because anormal GFP protein is fluorescent. This means that our GFP proteinwas not folded properly. Therefore, we can note that mutations inamino acids induced significant effects on protein structure, whichis directly related to protein function.

Our sequence had two mutated amino acids.First, we had Leucine mutated from Proline. The structures of the twoamino acids are shown below in figure 1 and figure 2.

Figure1: Structure of Proline Figure 2: Structure of Leucine

Eventhough both Proline and Leucine are classified as non-polar, andnon-charged amino acids, Proline contains a ring while Leucine is ina linear structure. In terms of the protein backbone formation,N-terminus of Proline is located in the ring, which will affect polypeptide bond formations due to its restricted conformation and sterichindrance. Such bulkily ring structure of Proline at N-terminus tendsto form 90-degree bond angle on peptide bond. On the other hand, thelinear structure of Leucine allows free rotation on peptide bond,making it more flexible for interactions. Such feature will greatlyaffect protein folding behavior of the amino acid sequences.

Another mutation took place at the 217thamino acid, changing Histidine to Arginine. The figures belowillustrate the chemical structures of the amino acids.

Figure3: Structure of Histidine Figure 4: Structure of Arginine

AlthoughHistidine contains a ring, while Arginine is in a linear structurewhich seemingly resembles the previous case, N-terminus of Histidineis not on the ring. Whether or not possessing a ring does notdirectly affect polypeptide bond formation. In this case, thedifferences in ionic charge is the key element that drives changes inprotein folding. Under physiological PH condition, Histidine isslightly basic, or sometimes considered neutral while Arginine is oneof the most basic amino acids. Histidine has a net charge of +1 atneutral environment. As such, Histidine and Arginine have differentdegrees of basicity leading them to react differently to thesurrounding environment and/or adjacent side chains resulting indifferent protein structures in outcome.