# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 3 Results
### Production of Recombinant Hsp60 and Pap1  
To assess the role of both Hsp60 and Pap1 during the _S. schenckii_-host interaction, we generated recombinant proteins in a prokaryotic system and used these polypeptides for the production of polyclonal antibodies against them. The open reading frames of the genes under study were amplified from _S. schenckii_ cDNA and cloned in the expression vector pCold I, generating pCold-_HSP60_ and pCold-_PAP1_, respectively. Gene expression was induced in _E. coli_ cells with isopropyl-\(\beta\)-D-1-thiogalactopyranoside and incubation at 15 \({}^{\circ}\)C. Several conditions for gene induction were tested in pilot experiments (not shown), and the best conditions were found to be 0.1 mM of the inductor and 20 h of incubation. Under these inducing conditions, two differentially expressed protein bands were observed in the cell \begin{table}
\begin{tabular}{c c c c c} \hline
**Protein** & **Gene** & **Coverage (\%)** & **\(q\)-Value** & **Andromeda Score** \\ \hline Chaperonin GroEL-like protein/heat shock protein 60 & SPSK\_01586 & 48.9 & 0 & 323 \\ Heat shock 70 kDa protein 1/8 & SPSK\_08625 & 47.6 & 0 & 323 \\ Uncharacterized protein & SPSK\_04236 & 22.2 & 0 & 323 \\ Uncharacterized protein & SPSK\_00848 & 21.3 & 0 & 323 \\ Uncharacterized protein & SPSK\_05930 & 19.8 & 0 & 323 \\ Glyceraldehyde-3-phosphate dehydrogenase & SPSK\_00294 & 38.3 & 0 & 322 \\ ATP synthase subunit beta & SPSK\_01537 & 54.7 & 0 & 307 \\ Elongation factor 1-alpha & SPSK\_06026 & 33.5 & 0 & 291 \\ GPI-anchored cell wall beta-1,3-endoglucanase EgIC & SPSK\_01694 & 21.2 & 0 & 260 \\ Aldehyde dehydrogenase (NAD+) & SPSK\_00262 & 28.4 & 0 & 244 \\ Large subunit ribosomal protein LP2 & SPSK\_03751 & 34.2 & 0 & 218 \\ Uncharacterized protein & SPSK\_01041 & 23.4 & 0 & 217 \\ AMPKK1\_CBM domain-containing protein & SPSK\_08400 & 27.6 & 0 & 197 \\ Molecular chaperone DnaK & SPSK\_03148 & 22.9 & 0 & 187 \\ Glucose-repressible protein & SPSK\_01322 & 64.8 & 0 & 182 \\ Peptideyl-propyl cis-trans isomerase & SPSK\_01963 & 24.2 & 0 & 172 \\ Uncharacterized protein & SPSK\_02764 & 50.3 & 0 & 158 \\ Large subunit ribosomal protein LP1 & SPSK\_05488 & 43.6 & 0 & 149 \\ Sphingolipid long chain base-responsive protein & SPSK\_05417 & 21.7 & 0 & 116 \\ Acetyltransferase component of pyruvate & SPSK\_07465 & 14.7 & 0 & 115 \\ dehydrogenase complex & & & & \\ Mismatched base pair and cruciform DNA & & & & \\ recognition protein & & & & \\ Aconitate hydratase, mitochondrial & SPSK\_00414 & 13.8 & 0 & 110 \\ Uncharacterized protein & SPSK\_06559 & 32.6 & 0 & 110 \\ Cytochrome c oxidase subunit 5b & SPSK\_03566 & 29.4 & 0 & 110 \\
2-phosphoglycerate dehydratase & SPSK\_03292 & 20.5 & 0 & 106 \\ Actin beta/gamma 1 & SPSK\_00108 & 15 & 0 & 104 \\ \hline \end{tabular}
\end{table} 
Table 1: Main identified proteins in peptidorhammonium by capillary liquid-chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry. homogenates, one of 65 kDa, corresponding to recombinant (r) Hsp60, and one of 32 kDa, corresponding to rPap1 (Figure 2A). Since the expression vector contained sequences for molecular tags that are part of the recombinant proteins, such as 6 xHis [56], it is likely that these extra sequences were responsible for the discrepancy between the experimental and predicted molecular weights of 62 kDa and 29 kDa for Hsp60 and Pap1, respectively. As a control, cells transformed with the empty pCold I vector did not show the differentially expressed protein bands (Figure 2A), suggesting that those protein bands of 65 and 32 kDa were the recombinant rHp60 and rPap1, respectively. Since both proteins contain the 6 xHis tag at the N-terminal end, protein purification was performed by affinity chromatography in a nickel-charged resin, followed by purification from SDS-PAGE gels, as reported for other proteins [56]. Following this strategy, one single protein band was observed in the purified fractions, with the corresponding 65 and 32 kDa protein bands for rHsp60 and rPap1, respectively (Figure 2B,C).