CHAPTER 12
A New Method for the Isolation of the C-Terminal
Peptide of Proteins by the Combination
of Selective Blocking of Proteolytic Peptides
and Cation Exchange Chromatography
L. J. GONZALEZ!, E. TORRES', Y. GARciA', L. H. BETANCOURT', G. MOYA2,
V. HUERTA, V. BESADA' and G. PADRON'
1
Introduction
The sequence information on the C-terminal end of proteins is highly appreciated. This information is used for the quality control of recombinant proteins to
ensure the correct transcription of the gen of interest and also provide an exact
idea about the C-terminal processing of the protein. In the case of unknown proteins, this information is very useful because it allows designing oligonucleotides
for the selective isolation of encoding gen and their further DNA sequencing.
The C-terminal sequencing has followed two separate trends, one of them has
been the development of methods that allow the direct sequencing of the Cterminal end of proteins, such as the development of the C-terminal sequencers,
the use of carboxypeptidases (CPases) and a chemical hydrolysis that mimics the
CPase degradation.
The protein chemists have devoted great efforts to the development of a Cterminal sequencer (Boyd et al. 1991, Bailey and Shively 1990, Shenoy et al. 1993,
Miller and Baley 1995) and considerable progress has been made in this direction, in fact, there is a C-terminal sequencer commercially available today. However, the performances of the C-terminal sequencers in terms of sensitivity, and
initial and repetitive yields are still inferior in comparison with the N-terminal
sequencers, which use the well-established Edman chemistry.
The use of CPases to release the amino acids from the C-terminus of the intact
proteins (Nguyen et al. 1995, Thiede et al. 1995) has an important limitation: Cpases are substrate-dependent and some proteins are resistant to CPase digestion.
The use of perfluorated organic acids and its corresponding anhydrides to
yield CPase mimetic degradation (Tsuguita et al. 1992, Takamoto et al. 1995)
works mainly in short peptides because, in the case of intact proteins, internal
degradation in several parts of the proteins can also take place.
These are the reasons why another trend has been followed in developing
strategies to isolate the C-terminal peptide of the protein from all the proteolytic
peptides, and subsequently perform its sequencing by well-established methods
such as Edman degradation or Mass Spectrometry (MS).
I Division of Physical-Chemistry and
2 Quality Control Division of the Center for Genetic Engineering and Biotechnology. P.O.Box 6162.
Havana Cuba.
A New Method for the Isolation of the C-Terminal
Peptide of Proteins by the Combination
of Selective Blocking of Proteolytic Peptides
and Cation Exchange Chromatography
L. J. GONZALEZ!, E. TORRES', Y. GARciA', L. H. BETANCOURT', G. MOYA2,
V. HUERTA, V. BESADA' and G. PADRON'
1
Introduction
The sequence information on the C-terminal end of proteins is highly appreciated. This information is used for the quality control of recombinant proteins to
ensure the correct transcription of the gen of interest and also provide an exact
idea about the C-terminal processing of the protein. In the case of unknown proteins, this information is very useful because it allows designing oligonucleotides
for the selective isolation of encoding gen and their further DNA sequencing.
The C-terminal sequencing has followed two separate trends, one of them has
been the development of methods that allow the direct sequencing of the Cterminal end of proteins, such as the development of the C-terminal sequencers,
the use of carboxypeptidases (CPases) and a chemical hydrolysis that mimics the
CPase degradation.
The protein chemists have devoted great efforts to the development of a Cterminal sequencer (Boyd et al. 1991, Bailey and Shively 1990, Shenoy et al. 1993,
Miller and Baley 1995) and considerable progress has been made in this direction, in fact, there is a C-terminal sequencer commercially available today. However, the performances of the C-terminal sequencers in terms of sensitivity, and
initial and repetitive yields are still inferior in comparison with the N-terminal
sequencers, which use the well-established Edman chemistry.
The use of CPases to release the amino acids from the C-terminus of the intact
proteins (Nguyen et al. 1995, Thiede et al. 1995) has an important limitation: Cpases are substrate-dependent and some proteins are resistant to CPase digestion.
The use of perfluorated organic acids and its corresponding anhydrides to
yield CPase mimetic degradation (Tsuguita et al. 1992, Takamoto et al. 1995)
works mainly in short peptides because, in the case of intact proteins, internal
degradation in several parts of the proteins can also take place.
These are the reasons why another trend has been followed in developing
strategies to isolate the C-terminal peptide of the protein from all the proteolytic
peptides, and subsequently perform its sequencing by well-established methods
such as Edman degradation or Mass Spectrometry (MS).
I Division of Physical-Chemistry and
2 Quality Control Division of the Center for Genetic Engineering and Biotechnology. P.O.Box 6162.
Havana Cuba.