Abstract
Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two primers (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 323-328 |
| Number of pages | 6 |
| Journal | Gene |
| Volume | 136 |
| Issue number | 1-2 |
| DOIs | |
| State | Published - Dec 22 1993 |
| Externally published | Yes |
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Keywords
- AIDS
- gene synthesis
- mutagenesis
- oligodeoxyribonucleotide
- Recombinant DNA
- retro viral protein, polymerase chain reaction
ASJC Scopus subject areas
- Genetics
Cite this
HIV1 integrase expressed in Escherichia coli from a synthetic gene. / Holler, Tod P.; Foltin, Susan K.; Ye, Qizhuang; Hupe, Donald J.
In: Gene, Vol. 136, No. 1-2, 22.12.1993, p. 323-328.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - HIV1 integrase expressed in Escherichia coli from a synthetic gene
AU - Holler, Tod P.
AU - Foltin, Susan K.
AU - Ye, Qizhuang
AU - Hupe, Donald J.
PY - 1993/12/22
Y1 - 1993/12/22
N2 - Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two primers (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.
AB - Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two primers (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.
KW - AIDS
KW - gene synthesis
KW - mutagenesis
KW - oligodeoxyribonucleotide
KW - Recombinant DNA
KW - retro viral protein, polymerase chain reaction
UR - http://www.scopus.com/inward/record.url?scp=0027733935&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027733935&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(93)90488-O
DO - 10.1016/0378-1119(93)90488-O
M3 - Article
C2 - 7916726
AN - SCOPUS:0027733935
VL - 136
SP - 323
EP - 328
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1-2
ER -