Essential Cell Biology 5th edition

Exploring Gene Function
361
Figure 10–34 Large amounts of a protein can be produced from
a protein-coding DNA sequence inserted into an expression
vector and introduced into cells. Here, a plasmid vector has
been engineered to contain a highly active promoter, which causes
unusually large amounts of mRNA to be produced from the inserted
protein-coding gene. Depending on the characteristics of the cloning
vector, the plasmid is introduced into bacterial, yeast, insect, or
mammalian cells, where the inserted gene is efficiently transcribed and
translated into protein.
yellow/orange color—could help to alleviate severe vitamin A deficiency,
which causes blindness in hundreds of thousands of children in the
developing world each year.
Even Rare Proteins Can Be Made in Large Amounts
Using Cloned DNA
One of the most important contributions of DNA cloning and genetic
engineering to cell biology is that they make it possible to produce any
protein, including the rare ones, in large amounts. Such high-level production
is usually accomplished by using specially designed vectors
known as expression vectors. These vectors include transcription and
translation signals that direct an inserted gene to be expressed at high
levels. Different expression vectors are designed for use in bacterial,
yeast, insect, or mammalian cells, each containing the appropriate regulatory
sequences for transcription and translation in these cells (Figure
10–34). The expression vector is replicated at each round of cell division,
so that the transfected cells in the culture are able to synthesize large
amounts of the protein of interest—sometimes comprising 1–10% of the
total cell protein. It is usually a simple matter to purify this protein away
from the other proteins made by the host cell.
This technology is now used to make large amounts of many medically
useful proteins, including hormones (such as insulin), growth factors, therapeutic
antibodies, and viral coat proteins for use in vaccines. Expression
vectors also allow scientists to produce many proteins of biological interest
in large enough amounts for detailed structural and functional studies
that were once impossible—especially for proteins that are normally
present in very small amounts, such as some receptors and transcription
regulators. Recombinant DNA techniques thus allow scientists to move
with ease from protein to gene, and vice versa, so that the functions of
both can be explored from multiple directions (Figure 10–35).
expression vector
promoter
sequence
CUT DNA WITH
RESTRICTION NUCLEASE
INSERT PROTEIN-
CODING DNA SEQUENCE
INTRODUCE
RECOMBINANT DNA
INTO CELLS
overexpressed protein
overexpressed
mRNA
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DETERMINE AMINO ACID
SEQUENCE OF A PEPTIDE FRAGMENT
USING MASS SPECTROMETRY
SEARCH DNA
DATABASE FOR
GENE SEQUENCE
SYNTHESIZE
DNA PRIMERS
AND CLONE BY PCR
STRUCTURAL AND BIOCHEMICAL
ANALYSES TO DETERMINE
THREE-DIMENSIONAL
CONFORMATION AND
ACTIVITY
PROTEIN
GENE or cDNA
MANIPULATE AND
INTRODUCE
ALTERED GENE INTO
CELLS OR ORGANISM
TO STUDY FUNCTION
OVEREXPRESS
AND PURIFY
PROTEIN
INTRODUCE INTO
E. coli OR OTHER
HOST CELL
INSERT PROTEIN-CODING
REGION OF GENE INTO
EXPRESSION VECTOR
Figure 10–35 Recombinant DNA techniques make it possible to move experimentally from gene to protein or from protein
to gene. A small quantity of a purified protein or peptide fragment is used to obtain a partial amino acid sequence, which is used to
search a DNA database for the corresponding nucleotide sequence. This sequence is used to synthesize DNA primers, which can be
used to clone the gene by PCR from a sequenced genome (see Figure 10–13). Once the gene has been isolated and sequenced, its
protein-coding sequence can be inserted into an expression vector to produce large quantities of the protein (see Figure 10−34), which
can then be studied biochemically or structurally. In addition to producing protein, the gene or DNA can also be manipulated and
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introduced into cells or organisms to study its function.