In the beginning, the bacterium was the centerpiece ofbiomanufacturing.

The first therapeutic proteins, insulin and growth hormone, wereproduced in these microorganisms. As the need to make larger andmore complex proteins increased, mammalian cells began to replacebacteria as the biomanufacturing cell of choice.

But while mammalian cells could correctly fold expressed foreignproteins and add the right sugar residues in the right order, they havenever been able to express recombinant proteins at levels as high asthose seen in bacteria. So, although no longer always at center stage,the bacterium has remained an integral part of the biomanufacturingscene.

With the increasing need to produce therapeutic proteins in largerquantities, production scientists are taking a second look at the highexpressing bacterial host. Unfortunately, the bacterium's expressionadvantage is largely neutralized during downstream processing stepsbecause it often produces denatured foreign proteins in an inactive,aggregated form in inclusion bodies that are expensive to process. Asa result, making bacteria secrete foreign proteins in soluble form hasbecome the goal. In essence, scientists are trying to exploit thebiology of the bacterial secretion process for biomanufacturingsuccess.

Chaperones, members of the heat shock protein family, areintimately involved in the processes of protein folding and assemblyin vivo. In the Feb. 14 issue of the Proceedings of the NationalAcademy of Sciences (PNAS), Kurt Amrein and his colleagues at theRoche Research Center, in Nutley, N.J., report on the use ofchaperones in a bacterial biomanufacturing process. Their article,"Purification and characterization of recombinant p50csk protein-tyrosine kinase from an Escherichia coli expression systemoverproducing the bacterial chaperones GroES and GroEL,"describes the increased production of a foreign protein in solubleform in bacteria using plasmids encoding these chaperone proteins.

Plasmids containing the coding sequences for the bacterialchaperones, GroES and GroEL, were introduced into an E. colistrain. These chaperones can increase the yield of correctly folded,soluble, active proteins both in vitro and in vivo. This E. coli strainalso had a vector containing the gene for human p50 csk, a proteintyrosine kinase of the Src family that mediates crucial intracellularregulatory phosphorylation events.

As reported in the PNAS article, overproduction of all three proteinsresulted in a dramatic increase in the solubility of protein-tyrosinekinase. More than half of the kinase was now found in the solubleprotein fraction. Using these methods, milligram amounts of activeenzyme were produced.

Amrein, senior author and molecular biologist, said, "This systemallowed us to produce enough kinase for characterization. We wereable to demonstrate that this kinase autophosphorylates, in contrastto what has been seen in kinase purified from eukaryotic cells."

Amrein told BioWorld Today that protein crystallization studies arebeing pursued. An overlay assay has been developed in which theprotein-tyrosine kinase is used as a probe for identifying substratesand inhibitors. The latter can potentially be developed as therapeuticagents for use in modulating the important intracellular signalingpathways controlled by this kinase.

Chaperones Facilitate Bacterial Biomanufacturing

The key improvement seen in using chaperones, said Amrein, was"the increased levels of solubility. Expression is not better per se, butthe solubility of expressed proteins is greatly improved. We regularlysee between 10 and 100 times the amount of expressed protein in asoluble form."

Amrein was unable to comment on Hoffmann-La Roche's patentposition in this area, but he did tell BioWorld Today, "We have usedthese chaperones for the expression of other proteins. Whether weuse the GroES/GroEL system or another chaperone, such as dnaK,just depends on which works most effectively. As a result, we have aseries of vectors that we try out with each protein."

He elaborated, saying, "Chaperones work often, but not always. It isnot predictable from one protein to the next. Each one must beevaluated individually. We do not yet know what the determiningcharacteristics are."

While chaperones improve the chances of achieving solubility andincreased yields, biomanufacturing strategies still need to bedeveloped empirically on a protein-by-protein basis. n

-- Chester Bisbee Special To BioWorld Today

(c) 1997 American Health Consultants. All rights reserved.