Purification of recombinant proteins

Proteins produced by recombinant DNA technology are usually purified by means identical to those available for purification of traditional non-recombinant proteins extracted directly from a natural producer source. In fact, purification 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 significantly upon the approach subsequently taken to purify the recombinant product: inclusion body formation and the incorporation of purification tags. The processes of inclusion body formation, recovery and recombinant protein renaturation have been considered in Chapter 5. Once the recombinant protein has been refolded, additional purification (if required) follows traditional lines.

Genetic engineering techniques also facilitate the incorporation of specific peptide or protein tags to the protein of interest. A tag is chosen that confers on the resultant hybrid protein some pronounced physicochemical characteristic, facilitating its subsequent purification. 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 purification of the hybrid protein by techniques such as ion-exchange, hydrophobic interaction or affinity 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 purified by cation-exchange chroma-tography. This approach has been used in the purification 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 purification by hydrophobic interaction chromatography. A purification tag consisting of polyhistidine may be employed to purify proteins by metal chelate chromatography.

Tags that facilitate protein purification by affinity 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 purified using a column containing immobilized IgG. Immunoaffin-ity purification may be employed if antibodies have been raised against the tag utilized.

Upon purification 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 specific protease or chemical cleavage method at the protein-tag fusion junction. Sequence-specific 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 specific 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 specific method chosen. Chemical methods, for example, must generally be carried out under harsh conditions, often requiring high temperatures or extremes of pH. Such conditions can have a detrimental effect on normal protein functioning. Proteolytic removal of tags is also often less than 100 per cent efficient. Selective cleavage of the tag must be followed by subsequent separation of the tag from the protein of interest. This may require a further chromatographic step.

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