- Poster Presentation
- Open Access
Point mutation of serine 179 in the human Prolactin (PRL) affects recombinant protein expression, folding and secretion, abolishes PRL nickel (II)-binding and increases heparin binding capacities
© Ueda et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Secretion Expression
- Soluble Aggregate
- Eukaryotic Expression System
- Human Prolactin
- Bioactivity Assay
S179D prolactin (S179D PRL) is a pseudophosphorylated form of human prolactin (PRL) which has inhibitory effects on tumor growth  and angiogenesis . The S179D PRL preparations used for these experiments consisted of properly refolded inclusion bodies (IB) from Escherichia coli . Trying to attain a better folded mutant, we used secretion expression based systems. However, single point mutations can affect protein periplasmic expression , and secretion from mammalian cells . We observed that upon a mutation of Serine 179 to an Aspartate, expression was nearly abolished when compared with PRL in E. coli periplasm, while the cytoplasmic product was more prone to proteolysis. Using eukaryotic cells we were able to produce preparations comparable to IBs in terms of bioactivity. We also demonstrated that this mutant had a higher affinity for heparin and lower binding capacity towards divalent metals (M (II)).
BL21 strain was used (figure 1B) without improvements for S179D PRL expression (table 2).
An eukaryotic expression system was chosen to successfully produce soluble, monomeric, recombinant S179D PRL.
Moreover S179D PRL had a decreased affinity towards Ni (II) ans Zn (II). On the other hand it had an increased affinity towards heparin.
Protein expression yield (μg/mL/OD) and final optical densities (OD600) of different strains with pL promoter.
Protein yield (μg/mL/OD)
1.3 ± 0.2
4.0 ± 0.3
1.9 ± 0.4
1.3 ± 0.2
BL21 codon plus
1.4 ± 0.3
1.0 ± 0.2
0.34 ± 0.03
3.8 ± 0.6
0.35 ± 0.5
1.3 ± 0.5
BL21 codon plus
0.40 ± 0.3
1.2 ± 0.1
- Xu X, Wu W, Williams V, Khong A, Chen YH, Deng C, Walker AM: Opposite effects of unmodified prolactin and a molecular mimic of phosphorylated prolactin on morphology and the expression of prostate specific genes in the normal rat prostate. Prostate. 2003, 54: 25-33. 10.1002/pros.10168.View ArticleGoogle Scholar
- Ueda E, Ozerdem U, Chen YH, Yao M, Huang KT, Sun H, Martins-Green M, Bartolini P, Walker AM: A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone. Endocr Relat Cancer. 2006, 13: 95-111. 10.1677/erc.1.01076.View ArticleGoogle Scholar
- Chen TJ, Kuo CB, Tsai KF, Liu JW, Chen DY, Walker AM: Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone. Endocrinology. 1998, 139: 609-16. 10.1210/en.139.2.609.Google Scholar
- Duenas M, Ayala M, Vazquez J, Ohlin M, Soderlind E, Borrebaeck CA, Gavilondo JV: A point mutation in a murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli . Gene. 1995, 158: 61-6. 10.1016/0378-1119(95)00077-J.View ArticleGoogle Scholar
- Sun Z, Lee MS, Rhee HK, Arrandale JM, Dannies PS: Inefficient secretion of human H27A-prolactin, a mutant that does not bind Zn2+. Mol Endocrinol. 1997, 11: 1544-51. 10.1210/me.11.10.1544.View ArticleGoogle Scholar
- Reyes LF, Sommer CA, Beltramini LM, Henrique-Silva F: Expression, purification, and structural analysis of (HIS)UBE2G2 (human ubiquitin-conjugating enzyme). Protein Expr Purif. 2006, 45: 324-8. 10.1016/j.pep.2005.08.018.View ArticleGoogle Scholar
- Bessette PH, Aslund F, Beckwith J, Georgiou G: Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci USA. 1999, 96: 13703-8. 10.1073/pnas.96.24.13703.View ArticleGoogle Scholar
- Makrides SC: Strategies for achieving high-level expression of genes in Escherichia coli . Microbiol Rev. 1996, 60: 512-38.Google Scholar
- Ellgaard L, Helenius A: Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol. 2003, 4: 181-91. 10.1038/nrm1052.View ArticleGoogle Scholar
- Ricard-Blum S, Feraud O, Lortat-Jacob H, Rencurosi A, Fukai N, Dkhissi F, Vittet D, Imberty A, Olsen BR, van der Rest M: Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations. J Biol Chem. 2004, 279: 2927-2936. 10.1074/jbc.M309868200.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd.