13. Outline the individual steps required to sequence a protein. What must be do

Question

13. Outline the individual steps required to sequence a protein. What must be done to apply the sequence before larger proteins can be sequenced? 14. Assume you have isolated a relatively abundant protein, and you want to obtain the amino acid sequence. You added dithiothreitol to the sample then did SDS-PAGE and found two bands A at 2.9 and B at 3.9 kD, You then cleaved the protein with chymotrypsin and found the following fragments: Band A CN Band B NLQNY GIVEQCCHKRCSEY DPTKM ACGVRGF RTTGHLCGKDLVNALY You then cleaved with Staphylococcus aureus V8 protease and found the following PTKM RTTGHLCGKD LVNALYIACGVRGFFYD Band B Band A GIVE YNLONYCN QCCHKRCSE What is the primary amino acid sequence f the protein?

Explanation / Answer

1: Splitting of the polypeptide:-

Many polypeptide chains are combined by a non covalent bond which is to be separated and purified in the first step

2: analysising the number of polypeptide in protein molecule :- The relation between number of moles of amino acid recidue and the molecular weight of protein determines the number of polypeptides.

3:- Cleavage of disulphide bonds :- many polypeptide chains are linked by disulphide bonds which are reduced to thiol and mercaptoethanol under the presence of hydrochloride.

4. Detecting the amino acid composition and calculating its molecular ratio.

5:- Arranging N terminal and C terminal of polypeptide chain .

6:- Polypeptides are again cleaved into smaller fragments of peptides .

7:- Determing the AA sequence for each peptide.

8:-Determining the sequence of peptide fragments , and determinating the position of disulphide bonds.

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