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Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli
KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.ORCID iD: 0000-0002-6979-0069
2005 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 2, 239-247 p.Article in journal (Refereed) Published
Abstract [en]

From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full-length protein, accumulation of the model fusion proteins Zb-MalE and Zb-MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed-batch cultivation.

Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb-MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure.

It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a "high" feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (<= 5 mu M), this formation is almost abolished while at a comparatively high concentration (>= 300 mu M) 50% of the total product accumulated was in the form of inclusion bodies.

Place, publisher, year, edition, pages
2005. Vol. 90, no 2, 239-247 p.
Keyword [en]
Escherichia coli, Fed-batch, Feed-rate, Inclusion bodies, Proteolysis, Solubility, Zb-MalE, Zb-MalE31
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-6751DOI: 10.1002/bit.20433ISI: 000227994700009OAI: oai:DiVA.org:kth-6751DiVA: diva2:11550
Note
QC 20100929Available from: 2007-02-02 Created: 2007-02-02 Last updated: 2010-10-08Bibliographically approved
In thesis
1. Methodology for high-throughput production of soluble recombinant proteins in Escherichia coli
Open this publication in new window or tab >>Methodology for high-throughput production of soluble recombinant proteins in Escherichia coli
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in Escherichia coli.

The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein.

The influence of feed rate on solubility and proteolysis was investigated using the lacUV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same.

The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter PmalK. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm.

Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. E.coli strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. 42 p.
Keyword
Escherichia coli, high-throughput methodology, recombinant protein production, solubility, productivity, quality, cultivation technology, parallel reactors
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4270 (URN)978-91-7178-561-9 (ISBN)
Presentation
2007-02-16, Sal FB54, AlbaNova universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20101112Available from: 2007-02-02 Created: 2007-02-02 Last updated: 2010-11-12Bibliographically approved
2. Impact of glucose feed rate on productivity and recombinant protein quality in Escherichia coli
Open this publication in new window or tab >>Impact of glucose feed rate on productivity and recombinant protein quality in Escherichia coli
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate.

The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, qp, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins.

By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation,

In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied.

In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the PmalK production system.

A novel fed-batch process based on the promoters for the universal stress proteins A and B (PuspA, PuspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 66 p.
Keyword
Biochemistry, Escherichia coli, recombinant protein production, fed-batch, feed profile, specific growth rate, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-115 (URN)91-7283-944-9 (ISBN)
Public defence
2005-02-04, Kollegiesalen, Valhallavägen 79, Stockholm, 10:00
Opponent
Supervisors
Note

QC 20101008

Available from: 2005-02-01 Created: 2005-02-01 Last updated: 2012-09-27Bibliographically approved

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