PCR Amplification of Staphylococcus Purine Biosynthetic Genes and Cloning into an Expression Vector

1
PCR Amplification of Staphylococcus Purine
Biosynthetic Genes and Cloning into an Expression
Vector

• Lori R. Schultz, Jeffrey D. Newman
• Lycoming College
• April 10, 1999

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Staphylococcus aureus An Evolving Superbug

• Resistant to multiple antibiotics
• Many strains of bacteria have become moderately
  resistant to vancomycin and other potent
  antibiotics
• Need to develop new antibiotics
• gene sequencing

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Staphylococcus aureus purY

• Entire genome not completely sequenced
• Obtained Staphylococcus aureus sequence from
  GenBank
• Retrieved purY sequence

4

Primer Design

• PCR primers incorporated Nde1 and EcoRV sites to
  allow cloning into the Nde1 and Sma1 site of the
  vector

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Cloning the Target Gene

• Amplified target gene by Polymerase Chain
  Reaction
• Purified the PCR product using QIAquick
  Purification Spin Columns
• Cut PCR product and vector with restriction
  enzymes

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Cloning the Target Gene

• Ligated PCR product into vector
• Transformed into E.coli
• isolated plasmids
• screened transformants by restriction enzyme
  digestion
• Confirmed clone by PCR

1 2 3 4 5 6 7 8
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Analyzing the Fusion Protein
Sequencing

• Intein Reverse Sequencing Primer

Intein reverse sequencing primer
5GCACCCATCTGCATTCTCCTTTTCATCATC 3
junction
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Expression of the Fusion Protein

• Addition of IPTG (isopropyl thiogalatoside) turns
  on T7 RNA polymerase gene allowing for expression
  of the fusion protein
• T7 promoter controls fusion gene expression
• Fusion protein was extracted using B-PER

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Expression of the Fusion Protein

• Fusion extract was analyzed by SDS-PAGE
• Maltose binding protein used as positive control
• Results provided a band for S. aureus purY
• Insoluble fraction contains the fusion protein

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Expression of the Fusion Protein

• PCR product confirms that only one copy of the
  gene is present

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Protein Purification

• Wash crude extract through chitin column
• Fusion protein binds to chitin column
• Flush column with DTT (1,4-Dithiothreitol)
• Target protein is released

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Protein Purification

• Lane 1 Protein Marker
• Lane 2 Crude extract from uninduced cells
• Lane 3 Crude extract from induced cells
• Lane 4 Clarified crude extract from induced
  cells
• Lane 5 Chitin column flow through
• Lane 6 Chitin column wash
• Lane 7 Quick DTT wash
• Lane 8 9 Fractions of eluted MBP
• Lane 10 SDS stripping of remaining proteins
  bound to chitin column