Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli

Kane, James F.

doi:10.1016/0958-1669(95)80082-4

Cited by 643 publications

(413 citation statements)

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“…Over-expression of recombinant protein in E. coli may be severely diminished if the codes of the ORF for the protein use "rare" codons that are infrequently used by E. coli (8)(9)(10)(11)(12). According to the online (http://nihserver.mbi.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Dai

Zhang

et al. 2009

Cell Mol Immunol

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HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37 o C. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni 2+ -affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues. Cellular & Molecular Immunology. 2009; 6(4):261-268.

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Section: Discussionmentioning

confidence: 99%

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Dai

Zhang

et al. 2009

Cell Mol Immunol

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“…The cDNA sequence for stefin B from GenBank (#LO3558) served as a scaffold for design. Whenever possible, codons present in highly expressed genes of E.coli (Kane, 1995) were selected to ensure good levels of expression. At the 5' end of the gene an EcoRI restriction site was added and at the 3' end two stop codons and a HindIII restriction site were included in order to make a direct cloning into pUC 18 (cloning vector) and pASK 40 (expression vector) possible.…”

Section: Synthesis Cloning Cassette Mutagenesis and Expression Of Hmentioning

confidence: 99%

Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of µ-Calpain

Díaz¹,

Größ²,

Assfalg-Machleidt³

et al. 2001

Biological Chemistry

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pose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold. Key words: Calpastatin / Kininogen / Papain / Stefin B / Surface plasmon resonance/ Temporary inhibition. IntroductionThe two ubiquitous calpains, µ-calpain and m-calpain, are intracellular, Ca 2+ -dependent cysteine proteinases that have been implicated in many important cellular functions and various pathologies (see Sorimachi et al., 1997;Carafoli and Molinari, 1998;Suzuki and Sorimachi, 1998 for recent reviews). They consist of distinct (yet homologous) large (L-)subunits (80 kDa) und a common small (S-)subunit (30 kDa). On the basis of sequence comparisons, the L-subunit has been predicted to contain four and the S-subunit two domains. Whereas the catalytic domain (domain II) of the L-subunit shows a weak sequence homology to papain, both the L-subunit and the S-subunit contain a Ca 2+ -binding calmodulin-like domain (CaMLD). Until recently, only three-dimensional structures of the L-CaMLD were known (Blanchard et al., 1997;Lin et al ., 1997), and hypotheses on the molecular mechanisms of activation and inhibition of calpains have been contradictory. Meanwhile, two groups have published crystal structures of the Ca 2+ -free, inactive form of m-calpain, revealing the molecular architecture of this multidomain protein (Hosfield et al., 1999;Strobl et al., 2000). In these structures, the catalytic domain (II) appears disrupted into two subdomains (IIa and IIb), explaining the inactivity of calpain in the absence of calcium. Activation should involve a 'fusion' of the two subdomains, leading to a functional papain-like catalytic domain (Hosfield et al., 1999;Strobl et al., 2000). As long as the structure of a Ca 2+ -activated calpain is not known, a number of questions concerning the molecular mechanisms of interaction with substrates and inhibitors remain open.Ca 2+ -activated µ-and m-calpain are controlled by a very specific intracellular protein inhibitor, calpastatin. Calpastatin contains four repeats of the inhibitory unit, each of which can inhibit calpain independently, but is not able to inhibit other cysteine proteinases of the papain superfamily (Maki et al ., 1987;Emori et al ., 1988 Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit -and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and ⌬L110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefinB by the corresponding KD2 sequence resulted in a calpain inhibitor of K i = 188 nM. Deletion of L110, which forms a ␤-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of -calpain 4-to 8-fold. All engineer...

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“…However, within a given genome, codon bias differs among individual genes according to codon position, suggesting that codon choice has functional consequences. For example, rare codons are enriched at the beginning of essential genes (7,8), and codon use strongly affects protein levels (9)(10)(11), especially at the N terminus (12). These observations suggest that codon use plays a poorly understood role in regulating protein expression.…”

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confidence: 99%

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Napolitano

Landon

Gregg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.codon choice | genome editing | recoded genomes

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Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli

Cited by 643 publications

References 22 publications

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Recombinant Expression of a Novel Human Transcriptional Repressor HMBOX1 and Preparation of Anti-HMBOX1 Monoclonal Antibody

Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of µ-Calpain

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

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