Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 160

a natural producer source. In fact, purifi cation of recombinant proteins can be somewhat more
straightforward, as high expression levels of the target protein can be attained. This increases the
ratio of target protein to contaminants.
Two features of recombinant production in particular can impact very signifi cantly upon the
approach subsequently taken to purify the recombinant product: inclusion body formation and
the incorporation of purifi cation tags. The processes of inclusion body formation, recovery and
recombinant protein renaturation have been considered in Chapter 5. Once the recombinant pro-
tein has been refolded, additional purifi cation (if required) follows traditional lines.
Genetic engineering techniques also facilitate the incorporation of specifi c peptide or protein
tags to the protein of interest. A tag is chosen that confers on the resultant hybrid protein some pro-
nounced physicochemical characteristic, facilitating its subsequent purifi cation. Such a molecule
is normally produced by fusing a DNA sequence that codes for the tag to one end of the genetic
information encoding the protein of interest. Tags that allow rapid and straightforward purifi ca-
tion of the hybrid protein by techniques such as ion-exchange, hydrophobic interaction or affi nity
chromatography have been designed and successfully employed.
Addition of a polyarganine (or polylysine) tag to the C-terminus of a protein confers on it a
strong positive charge. The protein may then be more readily purifi ed by cation-exchange chroma-
tography. This approach has been used in the purifi cation of various interferons and urogastrone
on a laboratory scale at least. Addition of a tag containing a number of hydrophobic amino acids
confers on the resultant molecule a strongly hydrophobic character, which allows its effective puri-
fi cation by hydrophobic interaction chromatography. A purifi cation tag consisting of polyhistidine
may be employed to purify proteins by metal chelate chromatography.
Tags that facilitate protein purifi cation by affi nity chromatography have also been developed.
The gene coding for protein A may be fused to the gene or cDNA encoding the protein of interest.
The resultant hybrid may be purifi ed using a column containing immobilized IgG. Immunoaffi n-
ity purifi cation may be employed if antibodies have been raised against the tag utilized.
Upon purifi cation of the hybrid protein it is necessary to remove the tag, as the tag itself will be
immunogenic. Removal of the tag is generally carried out by chemical or enzymatic means. This is
achieved by designing the tag sequence such that it contains a cleavage point for a specifi c protease
or chemical cleavage method at the protein-tag fusion junction. Sequence-specifi c proteases often
employed to achieve tag removal include the endopeptidases trypsin, factor Xa and enterokinase.
Exopeptidases, such as carboxypeptidase A, are also sometimes utilized. Generally speaking, en-
dopeptidases, which cleave internal protein peptide bonds, are used to remove long tags, whereas
exopeptidases are used most often to remove short tags. The exopeptidase carboxypeptidase A, for
example, sequentially removes amino acids from the C-terminus of a protein until it encounters a
lysine, arginine or proline residue. Chemical cleavage of specifi c peptide bonds relies on the use
of chemicals such as cyanogen bromide or hydroxylamine.
Although several methods exist that can achieve tag removal, most such methods suffer from some
inherent drawbacks. One essential prerequisite for any method is that the protein of interest must remain
intact after the cleavage treatment. The required protein, therefore, should not contain any peptide bonds
susceptible to cleavage by the specifi c method chosen. Chemical methods, for example, must generally
be carried out under harsh conditions, often requiring high temperatures or extremes of pH. Such con-
ditions can have a detrimental effect on normal protein functioning. Proteolytic removal of tags is also
often less than 100 per cent effi cient. Selective cleavage of the tag must be followed by subsequent sepa-
ration of the tag from the protein of interest. This may require a further chromatographic step.