Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System
By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed...
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| creator | Wang, Yalan Mao, Yuanhui Xu, Xiaodong Tao, Shiheng Chen, Hongying |
| description | By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells. |
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The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0145887</identifier><identifier>PMID: 26697848</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amino acids ; Analysis ; Animals ; Baculoviridae - genetics ; Baculovirus ; Baculoviruses ; Base Pairing ; Base Sequence ; Biology ; Codon - genetics ; Codon bias ; Codons ; Degradation ; E coli ; Escherichia coli ; Gene expression ; Gene sequencing ; Genomes ; Infections ; Insect cells ; Insects ; Laboratories ; Life sciences ; Luciferase ; Luciferases - metabolism ; Molecular Sequence Data ; mRNA stability ; Mutation ; Peptides ; Protein Biosynthesis ; Protein expression ; Protein folding ; Protein Processing, Post-Translational ; Protein Sorting Signals - genetics ; Protein structure ; Proteins ; Proteins - genetics ; Proteins - metabolism ; Proteolysis ; RNA Stability - genetics ; Secondary structure ; Secretion ; Signal peptides ; Spodoptera - cytology ; Spodoptera - genetics ; Spodoptera - virology ; Streptomyces ; Structural stability</subject><ispartof>PloS one, 2015-12, Vol.10 (12), p.e0145887-e0145887</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Wang et al 2015 Wang et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-11c59b6e5be6927876c0033ce56d4a53251cad6732da850d8eee42566e7b55183</citedby><cites>FETCH-LOGICAL-c692t-11c59b6e5be6927876c0033ce56d4a53251cad6732da850d8eee42566e7b55183</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689351/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689351/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26697848$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Krammer, Florian</contributor><creatorcontrib>Wang, Yalan</creatorcontrib><creatorcontrib>Mao, Yuanhui</creatorcontrib><creatorcontrib>Xu, Xiaodong</creatorcontrib><creatorcontrib>Tao, Shiheng</creatorcontrib><creatorcontrib>Chen, Hongying</creatorcontrib><title>Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells.</description><subject>Amino acids</subject><subject>Analysis</subject><subject>Animals</subject><subject>Baculoviridae - genetics</subject><subject>Baculovirus</subject><subject>Baculoviruses</subject><subject>Base Pairing</subject><subject>Base Sequence</subject><subject>Biology</subject><subject>Codon - genetics</subject><subject>Codon bias</subject><subject>Codons</subject><subject>Degradation</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Gene expression</subject><subject>Gene sequencing</subject><subject>Genomes</subject><subject>Infections</subject><subject>Insect cells</subject><subject>Insects</subject><subject>Laboratories</subject><subject>Life sciences</subject><subject>Luciferase</subject><subject>Luciferases - 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genetics</topic><topic>Baculovirus</topic><topic>Baculoviruses</topic><topic>Base Pairing</topic><topic>Base Sequence</topic><topic>Biology</topic><topic>Codon - genetics</topic><topic>Codon bias</topic><topic>Codons</topic><topic>Degradation</topic><topic>E coli</topic><topic>Escherichia coli</topic><topic>Gene expression</topic><topic>Gene sequencing</topic><topic>Genomes</topic><topic>Infections</topic><topic>Insect cells</topic><topic>Insects</topic><topic>Laboratories</topic><topic>Life sciences</topic><topic>Luciferase</topic><topic>Luciferases - metabolism</topic><topic>Molecular Sequence Data</topic><topic>mRNA stability</topic><topic>Mutation</topic><topic>Peptides</topic><topic>Protein Biosynthesis</topic><topic>Protein expression</topic><topic>Protein folding</topic><topic>Protein Processing, Post-Translational</topic><topic>Protein Sorting Signals - genetics</topic><topic>Protein structure</topic><topic>Proteins</topic><topic>Proteins - genetics</topic><topic>Proteins - metabolism</topic><topic>Proteolysis</topic><topic>RNA Stability - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Yalan</au><au>Mao, Yuanhui</au><au>Xu, Xiaodong</au><au>Tao, Shiheng</au><au>Chen, Hongying</au><au>Krammer, Florian</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-12-23</date><risdate>2015</risdate><volume>10</volume><issue>12</issue><spage>e0145887</spage><epage>e0145887</epage><pages>e0145887-e0145887</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>26697848</pmid><doi>10.1371/journal.pone.0145887</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Amino acids Analysis Animals Baculoviridae - genetics Baculovirus Baculoviruses Base Pairing Base Sequence Biology Codon - genetics Codon bias Codons Degradation E coli Escherichia coli Gene expression Gene sequencing Genomes Infections Insect cells Insects Laboratories Life sciences Luciferase Luciferases - metabolism Molecular Sequence Data mRNA stability Mutation Peptides Protein Biosynthesis Protein expression Protein folding Protein Processing, Post-Translational Protein Sorting Signals - genetics Protein structure Proteins Proteins - genetics Proteins - metabolism Proteolysis RNA Stability - genetics Secondary structure Secretion Signal peptides Spodoptera - cytology Spodoptera - genetics Spodoptera - virology Streptomyces Structural stability |
| title | Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System |
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