Question

Perform the following Expasy analyses. Provide screenshots of analyses for both translate and protparam calculations.

a) Translate the recAgene sequence to protein using “EXPASY: translate” tool. Copy and paste the RecA protein sequence into your answer sheet. (1 point)

Use “EXPASY Protparam” tool to calculate the molecular weight, number of amino acids and the theoretical pI of RecA protein as it will be expressed from the pET101-D-TOPO vector. (Clue: Copy the protein sequence into the EXPASY Protparam window. Remember to change “Met” to “M” so that the protparam tool would not read the “e” and “t” of Met as two different amino acids. Similarly remove “Stop” in the sequence when you do this analysis. Are there other regions (e.g., V5 epitope, His-tag) that are being included with RecA as part of cloning in pET 101-D? If so, include those sequences for the calculation as well). (1 point)

How many Cys, Tyr, and Trp are present in the protein? Do you anticipate problems if you use UV absorbance (A₂₈₀) for checking the protein concentration, based on the amino acid distribution? (Clue: Are there enough of the required type of amino acids present in RecA to use UV absorbance to measure protein concentration?). (1 point)

What is the extinction coefficient under fully reducing conditions (Note: extinction coefficient will be present towards the end of the EXPASY report.) (1 point)

Answer 1