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      Gene optimization mechanisms: A multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli

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          Abstract

          The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi-parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full-length human wt and sequence-optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E. coli. Fluorescence-based quantification revealed increased protein yields for 70% of in vivo expressed optimized genes compared to the wt DNA sequences and also resulted in increased amounts of protein that can be purified. The improvement in transgene expression correlated with higher mRNA levels in our analyzed examples. In all cases tested, expression levels using wt genes in tRNA-supplemented bacterial strains were outperformed by optimized genes expressed in non-supplemented host cells.

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          Most cited references36

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          Protein production and purification.

          In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
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            The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization

            One of the main advantages of de novo gene synthesis is the fact that it frees the researcher from any limitations imposed by the use of natural templates. To make the most out of this opportunity, efficient algorithms are needed to calculate a coding sequence, combining different requirements, such as adapted codon usage or avoidance of restriction sites, in the best possible way. We present an algorithm where a “variation window” covering several amino acid positions slides along the coding sequence. Candidate sequences are built comprising the already optimized part of the complete sequence and all possible combinations of synonymous codons representing the amino acids within the window. The candidate sequences are assessed with a quality function, and the first codon of the best candidates’ variation window is fixed. Subsequently the window is shifted by one codon position. As an example of a freely accessible software implementing the algorithm, we present the Mr. Gene web-application. Additionally two experimental applications of the algorithm are shown.
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              Advances in Escherichia coli production of therapeutic proteins.

              Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically sensitive products as insulin and bovine growth hormone. Although significant progress has been made in transcription, translation and secretion, one of the major challenges is obtaining the product in a soluble and bioactive form. Recent progress in oxidative cytoplasmic folding and cell-free protein synthesis offers attractive alternatives to standard expression methods.
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                Author and article information

                Journal
                Protein Sci
                pro
                Protein Science : A Publication of the Protein Society
                Wiley Subscription Services, Inc., A Wiley Company
                0961-8368
                1469-896X
                July 2010
                03 May 2010
                : 19
                : 7
                : 1312-1326
                Affiliations
                [1 ]simpleQIAGEN GmbH, QIAGEN Strasse 1 Hilden 40724, Germany
                [2 ]simpleRiNA GmbH, Takustrasse 3 Berlin 14195, Germany
                [3 ]simpleGeneart AG, BioPark, Joseph-Engert-Str. 11 Regensburg 93053, Germany
                [4 ]simpleMolecular Microbiology and Gene Therapy Unit, Institute of Medical Microbiology and Hygiene, University of Regensburg Regensburg 93053, Germany
                Author notes

                Additional Supporting Information may be found in the online version of this article.

                Conflict of Interest: The authors declare competing financial interests: Geneart AG performs gene design optimization as a free service with the genes that it sells. The authors also declare competing interests in the form of a pending relevant PCT patent application, no. WO04059556. FS owns stocks and stock options in QIAGEN GmbH.

                Barbara Maertens and Anne Spriestersbach contributed equally to this work.

                Grant sponsor: Bundesministerium für Bildung und Forschung (BMBF); Grant numbers: 0313965A, 0313965B, 0313850.

                * Correspondence to: Frank Schäfer, QIAGEN Strasse 1, Hilden 40724, Germany. E-mail: frank.schaefer@ 123456qiagen.com
                Article
                10.1002/pro.408
                2970903
                20506237
                dc337804-e140-43d5-8890-b44b86f6b701
                Copyright © 2010 The Protein Society
                History
                : 22 January 2010
                : 21 April 2010
                : 21 April 2010
                Categories
                Article

                Biochemistry
                fluorescence-based quantification,codon usage,recombinant proteins,synthetic genes,gene optimization,heterologous protein expression

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