5.2 Make Your Own Enzymes: Some Guidelines
for the Non-specialist
While basic knowledge of molecular biology is necessary to allow designing and
performing cloning, the corresponding theoretical background and most standard
protocols can be learnt rapidly by non-specialists. Provided the enzyme of interest
is neither particularly exotic nor resilient, any researcher with minimal equipment
(molecular biology kits, PCR machines and incubators are of advantage) can obtain
a first sample of protein from scratch within few weeks.
Accessing protein and gene sequence information
In case the enzyme of interest has been already characterized and (at least) its amino
acid sequence is known, a search in a protein database (most comprehensive one
being UniprotKB) can directly grant access to the peptide sequence. Back-translation
using online bioinformatics tools (numerous and freely accessible) delivers a possible
nucleic acid sequence, i.e. the gene, which codes for the protein of interest.
For most known protein sequences, the encoding gene is available directly in the
protein database (ID card of the protein) or the genetic information can be retrieved
from gene databases (GenBank, EMBL).
From the gene to the protein
With the gene of interest at hand, the next step involves decision on the host
organism to be used for protein expression. Typically, and for most standard
enzymes, the bacterium Escherichia coli is the workhorse. The gene can be ordered
from commercial vendors, who also offer complementary codon usage optimization, i.e. the gene sequence will be synthesized using the most frequently used
codon for each amino acid for a given host organism. In order to facilitate protein
purification, you may add a tag to the protein sequence.
Once an appropriate expression vector for gene cloning has been chosen, the
gene of interest is inserted in the plasmid through ligation and the recombinant
DNA product thus obtained can be transferred to the selected host (transformation).
Common commercial vectors are the pET vectors.
Finally, the recombinant protein is obtained by over-expression through growing
a cell culture of the host. Typically, IPTG is used as inducer of protein expression.
In case the protein forms inclusion bodies, is misfolded or turns out to be toxic
towards the host, ask your friends at the molecular biology department for troubleshooting strategies.
Several options for the use of this biocatalyst are available:
• Whole recombinant (E. coli) host cells can be used as such in a non-fermenting
(resting) state, particularly for preparative-scale biotransformations. In this case,
control experiments are necessary to ensure the absence of disturbing background activity by ‘empty’ host cells.
• Host cells are lysed to liberate the enzyme of interest.
• The enzyme of interest is purified after cell lysis, which is greatly facilitated by
an affinity tag.
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5 Appendix
for the Non-specialist
While basic knowledge of molecular biology is necessary to allow designing and
performing cloning, the corresponding theoretical background and most standard
protocols can be learnt rapidly by non-specialists. Provided the enzyme of interest
is neither particularly exotic nor resilient, any researcher with minimal equipment
(molecular biology kits, PCR machines and incubators are of advantage) can obtain
a first sample of protein from scratch within few weeks.
Accessing protein and gene sequence information
In case the enzyme of interest has been already characterized and (at least) its amino
acid sequence is known, a search in a protein database (most comprehensive one
being UniprotKB) can directly grant access to the peptide sequence. Back-translation
using online bioinformatics tools (numerous and freely accessible) delivers a possible
nucleic acid sequence, i.e. the gene, which codes for the protein of interest.
For most known protein sequences, the encoding gene is available directly in the
protein database (ID card of the protein) or the genetic information can be retrieved
from gene databases (GenBank, EMBL).
From the gene to the protein
With the gene of interest at hand, the next step involves decision on the host
organism to be used for protein expression. Typically, and for most standard
enzymes, the bacterium Escherichia coli is the workhorse. The gene can be ordered
from commercial vendors, who also offer complementary codon usage optimization, i.e. the gene sequence will be synthesized using the most frequently used
codon for each amino acid for a given host organism. In order to facilitate protein
purification, you may add a tag to the protein sequence.
Once an appropriate expression vector for gene cloning has been chosen, the
gene of interest is inserted in the plasmid through ligation and the recombinant
DNA product thus obtained can be transferred to the selected host (transformation).
Common commercial vectors are the pET vectors.
Finally, the recombinant protein is obtained by over-expression through growing
a cell culture of the host. Typically, IPTG is used as inducer of protein expression.
In case the protein forms inclusion bodies, is misfolded or turns out to be toxic
towards the host, ask your friends at the molecular biology department for troubleshooting strategies.
Several options for the use of this biocatalyst are available:
• Whole recombinant (E. coli) host cells can be used as such in a non-fermenting
(resting) state, particularly for preparative-scale biotransformations. In this case,
control experiments are necessary to ensure the absence of disturbing background activity by ‘empty’ host cells.
• Host cells are lysed to liberate the enzyme of interest.
• The enzyme of interest is purified after cell lysis, which is greatly facilitated by
an affinity tag.
418
5 Appendix