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Methodology for high-throughput production of soluble recombinant proteins in Escherichia coli
KTH, School of Biotechnology (BIO).
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 [en]
Escherichia coli, high-throughput methodology, recombinant protein production, solubility, productivity, quality, cultivation technology, parallel reactors
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-4270ISBN: 978-91-7178-561-9 (print)OAI: oai:DiVA.org:kth-4270DiVA: diva2:11553
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
List of papers
1. Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli
Open this publication in new window or tab >>Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli
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.

Keyword
Escherichia coli, Fed-batch, Feed-rate, Inclusion bodies, Proteolysis, Solubility, Zb-MalE, Zb-MalE31
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-6751 (URN)10.1002/bit.20433 (DOI)000227994700009 ()
Note
QC 20100929Available from: 2007-02-02 Created: 2007-02-02 Last updated: 2017-12-14Bibliographically approved
2. Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli
Open this publication in new window or tab >>Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli
Show others...
2005 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 68, no 1, 82-90 p.Article in journal (Refereed) Published
Abstract [en]

The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

Keyword
fed-batch production, cultivations, expression, promoters, fusion, system
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-6752 (URN)10.1007/s00253-004-1855-4 (DOI)000230859900013 ()2-s2.0-23944489637 (Scopus ID)
Note

QC 20101008

Available from: 2007-02-02 Created: 2007-02-02 Last updated: 2017-12-14Bibliographically approved
3. Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control
Open this publication in new window or tab >>Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control
2008 (English)In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, no 1, 11-20 p.Article in journal (Refereed) Published
Abstract [en]

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h(-1), respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.

Keyword
fed-batch technique, acetate formation, high cell density, recombinant product formation, phosphotransferase system, PTS mutations
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-8368 (URN)10.1007/s00449-007-0144-x (DOI)000251649100003 ()2-s2.0-37249015436 (Scopus ID)
Note
QC 20100902. Uppdaterad från Accepted till Published (20100902)Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2017-12-14Bibliographically approved

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