Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli
Abstract Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thou...
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ftdoajarticles:oai:doaj.org/article:e90df5456ba44ad6aa745cd2cedbc3d3 2023-05-15T15:15:33+02:00 Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli Chene Arnaud Ahuja Sanjay Flick Kirsten Bejarano Maria Chen Qijun 2004-12-01T00:00:00Z https://doi.org/10.1186/1475-2875-3-50 https://doaj.org/article/e90df5456ba44ad6aa745cd2cedbc3d3 EN eng BMC http://www.malariajournal.com/content/3/1/50 https://doaj.org/toc/1475-2875 doi:10.1186/1475-2875-3-50 1475-2875 https://doaj.org/article/e90df5456ba44ad6aa745cd2cedbc3d3 Malaria Journal, Vol 3, Iss 1, p 50 (2004) Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2004 ftdoajarticles https://doi.org/10.1186/1475-2875-3-50 2022-12-31T13:58:39Z Abstract Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Malaria Journal 3 1 50 |
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Directory of Open Access Journals: DOAJ Articles |
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language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
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Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Chene Arnaud Ahuja Sanjay Flick Kirsten Bejarano Maria Chen Qijun Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
Abstract Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons. |
format |
Article in Journal/Newspaper |
author |
Chene Arnaud Ahuja Sanjay Flick Kirsten Bejarano Maria Chen Qijun |
author_facet |
Chene Arnaud Ahuja Sanjay Flick Kirsten Bejarano Maria Chen Qijun |
author_sort |
Chene Arnaud |
title |
Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli |
title_short |
Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli |
title_full |
Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli |
title_fullStr |
Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli |
title_full_unstemmed |
Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli |
title_sort |
optimized expression of plasmodium falciparum erythrocyte membrane protein 1 domains in escherichia coli |
publisher |
BMC |
publishDate |
2004 |
url |
https://doi.org/10.1186/1475-2875-3-50 https://doaj.org/article/e90df5456ba44ad6aa745cd2cedbc3d3 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Malaria Journal, Vol 3, Iss 1, p 50 (2004) |
op_relation |
http://www.malariajournal.com/content/3/1/50 https://doaj.org/toc/1475-2875 doi:10.1186/1475-2875-3-50 1475-2875 https://doaj.org/article/e90df5456ba44ad6aa745cd2cedbc3d3 |
op_doi |
https://doi.org/10.1186/1475-2875-3-50 |
container_title |
Malaria Journal |
container_volume |
3 |
container_issue |
1 |
container_start_page |
50 |
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1766345923373301760 |