User:Jennifer Taylor/Sandbox 4

4Q7Q is a protein found in Chitinophaga pinensis, a soil bacterium in the sphingobacterial family. Its structure has been previously characterized and exists in Protein Data Bank. Its function, however, has not.

After structural and sequential analysis via various databases including BLAST, Pfam, Dali, PyMOL, and ProMOL, we initially predicted that 4Q7Q is a hydrolase. More specifically, we hypothesized that it was a lipase, an enzyme that can hydrolyze lipids to form fatty acids and a glycerol molecule.

4Q7Q's Structure

4Q7Q is composed of two chains, one chain can be seen . The colors indicate the translation direction of the peptide sequence from the N to C terminus; red represents the N-terminus while dark blue represents the C-terminus. Based on this structural model, we can see that 4Q7Q is an alpha-beta superfold; there are beta sheets (represented by the straighter strands) sandwiched between the alpha helices (represented by the coiled strands).

In silico Analysis

We initially analyzed 4Q7Q through the protein structure databases BLAST, Pfam, and Dali. Our top hit was 4M8K, a GDSL-like lipase. Through BLAST, we found that 4M8K and 4Q7Q had a 36% sequence identity, with an E value of 0.002, indicating that it is a significant match. Since we can use the principle of homology to predict the function of an unknown protein, we first hypothesized that 4Q7Q was too a lipase.

We also performed further analysis in PyMOL and ProMOL which involved the homology of active sites. Top hits included 3LIP, a lipase found in Burkholderia cepacia, 1TAH, a lipase found in Burkholderia glumae, and 1BWR, a hydrolase found in Bos taurus.

Bacterial Transformation

Before characterizing the function of 4Q7Q, we first needed to synthesize the protein through first transcribing 4Q7Q's DNA to amplify it and then translating it to express it. First, 4Q7Q's DNA was transcribed using its expression vector, the plasmid pMCS573. Since transformation must occur within a cell, the plasmid was transformed into DH5a cells using protocol from New England Biolabs. The cells were then spread on plates containing LB and ampicillin. (*explain) Then, the DH5a cells were lysed and the plasmid was purified.

However, though DH5a cells are E. coli cells specifically engineered to maximize the efficiency of transformations, they do not contain T7 polymerase, which is essential for protein expression (**explain more). Therefore, the purified plasmid underwent another bacterial transformation into BL21 (DE3) cells that do contain T7 polymerase using protocol from New England Biolabs.

Protein Expression

Protein Purification

pNPB Lipase Assay

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