Molecular Biotechnology: Principles and Applications - Glick, B. R., Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Enhancing Secretion Efficiency

The stability of Proteins encoded by cloned genes depends on their cellular localization. For instance, recombinant proinsulin proves to be approximately 10 times more stable when secreted (exported) into the periplasm (the space between the plasma and outer membranes) rather than remaining in the Cytoplasm. In addition, proteins secreted into the periplasm or the culture medium are easier to purify.

Typically, Protein Transport Across The Cell membrane is mediated by N-terminal Amino acid sequences known as signal Peptides (signal sequences or leader peptides). Occasionally, a protein can be rendered secretable by attaching The nucleotide sequence responsible for signal Peptide Synthesis to its structural Gene. However, the mere presence of a signal peptide does not guarantee efficient secretion. Furthermore, E. coli and other Gram-negative microorganisms are usually unable to secrete proteins into the surrounding medium due to the presence of an outer membrane. There are at least two ways to overcome this limitation: the first is to use Gram-positive prokaryotes or eukaryotes lacking an outer membrane, and the second is to engineer Gram-negative Bacteria capable of secreting proteins into the medium.

If the fusion of a target gene with a DNA fragment encoding a signal peptide does not lead to efficient protein secretion, alternative strategic approaches must be employed. One such approach, successfully applied to interleukin-2, was based on fusing the interleukin-2 gene with the gene encoding the full-length maltose-binding protein precursor (rather than just its signal sequence) and separating these genes with a DNA segment encoding a recognition site for factor X_a. When this chimeric gene was inserted into a plasmid vector and used to transform E. coli, large amounts of the chimeric protein were detected in the host cell periplasm. Subsequent Treatment with factor X_a yielded functional interleukin-2.

According to one study, the secretion of many heterologous proteins in E. coli depends on the expression level of the corresponding genes. Foreign proteins synthesized at the highest rates are not necessarily secreted with equal efficiency. Sometimes, the intensive synthesis of a foreign protein overloads the secretory machinery and blocks it. Therefore, if guaranteed secretion of a target protein is required, one can attempt to lower the expression level of the corresponding genes.

Certain Gram-negative bacteria secrete a protein called bacteriocin into the medium. It activates phospholipase A located in the inner membrane of the bacterial cell, thereby permeabilizing both the inner and outer membranes and releasing several cytosolic and periplasmic proteins into the culture medium. Thus, the bacteriocin gene can be inserted into a plasmid under the control of a strong regulated promoter, used to transform E. coli Cells, and thereby render them permeable. Alternatively, if E. coli strains already harbor the bacteriocin gene, they can be transformed with a second plasmid carrying the gene of interest fused to a nucleotide sequence encoding a signal peptide. If both genes are placed under the control of the same promoter, they can be induced simultaneously, resulting in the secretion of the cloned gene product into the medium.

When secreted foreign proteins are produced in E. coli in excessive amounts, precursor Processing is very often incomplete: approximately half of the secreted proteins retain their leader sequence, while the other half are fully processed into their mature form. This may be due to a shortage of certain proteins involved in the secretion pathway. To increase the proportion of processed proteins in such situations, one can attempt to upregulate the genes responsible for synthesizing the limiting Components of the secretory apparatus. To test this hypothesis, the following experiments were conducted. A plasmid carrying the prlA4 and secE genes, which encode essential components of the molecular machinery responsible for the physical translocation of proteins across the membrane, was introduced into E. coli cells. Following this reinforcement of the host cell secretory apparatus, the proportion of the recombinant protein (the cytokine interleukin-6) secreted in its mature form increased from 50% to over 90%.

Aspergillus Fungi secrete large amounts of Enzymes into the medium and are widely used for their industrial production. In one study, the human interferon gene was fused with a secretion signal peptide gene, and this construct was placed under the control of the starch-inducible glucoamylase promoter from Aspergillus nidulans. Upon addition of starch to the medium containing the transformed Aspergillus nidulans cells, the yield of secreted human interferon reached 1 mg per liter, which corresponds to approximately 5% of the total secreted cellular protein. This study demonstrates that Gene Expression modulation strategies developed for E. coli can also be successfully applied to other biological systems.