Science Homework Help

I have answer this questions but I am not sure if that is right so I want some one to fix and improve my answer if needed! please check fille call Homework 2 for more information for the ass

A fragment of human progranulin (PGRN, Homo sapiens) is overproduced in several human cancers.The fragment’s amino acid sequence is

GAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL

The fragment contains six repeats called granulins with the sequence pattern of

XXXCX…XCX…XCCX…XCCX…XCCX…XCCX…XCXXXXXX

where X is any amino acid residue, C is cysteine, … stands for one to several arbitrary amino acid residues in a row.

You would like to use one of the repeats (specifically, human granulin A, or hGrnA) as a protein marker for early cancer detection. As a part of your efforts to develop the diagnostic kit, you would like to quantify molecular interactions (affinity) between hGrnA and its cellular receptor (a protein known to bind hGrnA and trigger cell division upon binding).hGrnA is the third repeat out of the six if counting from the N-terminus.

To produce hGrnA you have decided to sub-clone it into the Novagen expression vector pET-21a between restriction sites HindIII and XhoI.The recombinant DNA construct must have the following specifications: a) The enterokinase cleavage site DDDDK to ensure that the N-terminal amine of the recombinant hGrnA after treatment with enterokinase is free and available for binding to the receptor; b) An arginine residue (R in one-letter code, Arg in three letter code) immediately after the last residue of hGrnA to improve the construct solubility, and c) a C-terminal hexahistidine tag after (His-tag, His₆) to enable hGrnA purification.

Your assignment is:

• Identify the amino acid sequence of hGrnA for subcloning.
• Design the DNA sequence encoding hGrnA optimized for Escherichia coli expression (use any K12 E. coli strain) and amenable for cloning between HindIII and XhoI restriction sites.Make sure that the back-translated optimized DNA sequence of hGrnA does not contain restriction sites HindIII and XhoI.Use genomes.urv.es/OPTIMIZER as shown during the class (codon usage HEG, method “guided random”).
• Design two primers (one forward and one reverse) with melting temperatures Tm equal to approximately 60-62°C for subcloning the synthetic gene into the pET-21a plasmid and creating a recombinant DNA construct that will satisfy the specifications a)-c).

The report must be submitted as a Word file.