You have been given an unknown peptide by a colleague and asked to provide an amino

acid sequence. Using the experimental data shown below, deduce the peptide sequence giving
the proper order of the amino acids and including the position of any disulfide bonds. Using
your best deduced sequence, provide the complete chemical structure for the unknown
peptide (ie. before hydrolysis). You may not be able to provide the sequence for ALL amino
acids. Indicate which amino acids for which you are uncertain.
(NOTE: Amino acids given in peptide fragments are the result of the complete hydrolysis
with HCl + HEAT and are given in alphabetical order, not in order of primary structure).

Expt. 1: Chymotrypsin digestion of the unknown peptide resulted in 3 peptide fragments.

The peptides were separated, treated with β-mercaptoethanol and hydrolyzed overnight in
HCl. The following results were obtained:
Frag. 1 (after hydrolysis): Arg, Asn, Glu, Leu, Thr
Frag. 2 (after hydrolysis): Asp, Phe
Frag. 3 (after hydrolysis): Cys, His, Lys, Met, Trp
NOTE: β-Mercaptoethanol treatment of individual fragments above did not result in multiple
fragments.

Expt. 2: Trypsin digestion of the unknown peptide resulted in 3 fragments that were
separated, treated with β-mercaptoethanol and hydrolysed overnight in HCl. The following
results were obtained:
Frag. 4 (after hydrolysis): Arg, Cys, Leu, Trp
Frag. 5 (after hydrolysis): Asn, Glu, Thr
Frag. 6 (after hydrolysis): Asp, His, Lys, Met, Phe
NOTE: β-Mercaptoethanol treatment of individual fragments above did not result in multiple
fragments.

Expt. 3: Cyanogen Bromide treatment resulted in:

Frag. 7 (after hydrolysis): Arg, Asn, Cys, Glu, His, Leu, Lys, Thr. Trp
Frag. 8 (after hydrolysis): Asp, Met, Phe
NOTE: β-Mercaptoethanol treatment of individual fragments above did not result in multiple
fragments.

Expt. 4: Carboxypeptidase released Glu

You have been given an unknown peptide by a colleague and asked to provide an amino
acid sequence. Using the experimental data shown below, deduce the peptide sequence giving
the proper order of the amino acids and including the position of any disulfide bonds. Using
your best deduced sequence, provide the complete chemical structure for the unknown
peptide (ie. before hydrolysis). You may not be able to provide the sequence for ALL amino
acids. Indicate which amino acids for which you are uncertain.
(NOTE: Amino acids given in peptide fragments are the result of the complete hydrolysis
with HCl + HEAT and are given in alphabetical order, not in order of primary structure).

Expt. 1: First cycle of Edman’s degradation on the unknown peptide results in Proline.

Expt. 2: Trypsin digestion of the unknown peptide resulted in 2 fragments that were
separated, treated with -mercaptoethanol and hydrolyzed overnight in HCl. The following
results were obtained:
Frag. 1 (after hydrolysis): Arg, Cys, His, Ile, Lys, Met, Phe, Pro, Thr, Trp, Tyr
Frag. 2 (after hydrolysis): Glu, Leu, Lys, Met, Trp
NOTE: Treatment of Frag. 1 with -mercaptoethanol gave:
Frag. 1a (after hydrolysis): Arg, Cys, Pro, Thr, Trp
Frag. 1b (after hydrolysis): Cys, His, Ile, Lys, Met, Phe, Tyr

Expt. 3: Chymotrypsin digestion of the unknown peptide resulted in 2 fragments that were
separated, treated with -mercaptoethanol and hydrolysed overnight in HCl. The following
results were obtained:
Frag. 3 (after hydrolysis): Glu, Ile, Leu, Lys, Met, Trp
Frag. 4 (after hydrolysis): Cys, His, Met, Pro, Trp, Tyr
Frag. 5 (after hydrolysis): Lys
Frag. 6 (after hydrolysis): Arg, Phe, Thr

NOTE: Treatment of Frag. 4 with -mercaptoethanol gave:

Frag. 4a (after hydrolysis): Cys, Pro, Trp
Frag. 4b (after hydrolysis): Cys, His, Met, Tyr

Expt. 4: Cyanogen Bromide treatment resulted in:

Frag. 7 (after hydrolysis): Arg, Cys, His, Met, Phe, Pro, Thr. Trp
Frag. 8 (after hydrolysis): Ile, Leu, Lys, Met, Tyr
Frag. 9 (after hydrolysis): Glu, Lys, Trp
NOTE: -Mercaptoethanol treatment of individual fragments from Expt 4 did not result in
multiple fragments.

Reagent Cleavage Site

Trypsin C-side of Lys, Arg
Chymotrypsin C-side of Phe, Tyr or Trp
Cyanogen Bromide C-side of Met
Carboxypeptidase C-terminal amino acid

A schematic of the vector p7012 is shown. The restriction enzymes listed cut only where indicated; they do
not cut anywhere else in the vector or insert.
b) In which of the strategies would Gene W be inserted into the vector in only one direction?