Part:BBa_K5246031

HB HfsH Deacetylase, 6xHis tag for purification

Usage and Biology

TBA

This part also has a non his-tagged variant BBa_K5246020.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 385
  
• 1000
  COMPATIBLE WITH RFC[1000]

Hirschia baltica

We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.

After SDS-PAGE gel analysis, we concluded that we successfully expressed HfsG, HfsH, HfsK, and HfsL proteins from H. baltica.

HfsH is visible on the right side of the gel (Fig. 1).

Table 1. H. baltica protein sizes in kDa

Protein Name	Size (kDa)
HfsG	37
HfsH	29
HfsJ	41
HfsK	28
HfsL	36

Fig. 1. 12% SDS-PAGE analysis of H. baltica in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

References

1. Wan, Z. et al. (2013a) ‘The adhesive and cohesive properties of a bacterial polysaccharide adhesin are modulated by a deacetylase’, Molecular Microbiology, 88(3), pp. 486–500. doi:10.1111/mmi.12199.
2. Toh, E., Kurtz, Harry D. and Brun, Y.V. (2008) ‘Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps’, Journal of Bacteriology, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.
3. Liu, Q. et al. (2022) ‘The screening and expression of polysaccharide deacetylase from caulobacter crescentus and its function analysis’, Biotechnology and Applied Biochemistry, 70(2), pp. 688–696. doi:10.1002/bab.2390.