Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 18;11(12):1916-1921.
doi: 10.18240/ijo.2018.12.06. eCollection 2018.

Recombination and identification of human alpha B-crystallin

Rui Wang  1 , Ze-Hua Chen  1 , Yi Wang  2 , Hou-Bin Huang  1 , Si-Jun Fan  3 , Lan-Lan Chen  1
Affiliations

Recombination and identification of human alpha B-crystallin

Rui Wang et al. Int J Ophthalmol. .

Abstract

Aim: To recombine the human alpha B-crystallin (αB-crystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin.

Methods: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αB-crystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli (E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.

Results: Compared with the gene bank (GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity.

Conclusion: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin is successfully constructed, and the recombinant human αB-crystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.

Keywords: gene; human alpha B-crystallin; identification; recombination; vector construction.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Gel electrophoresis following Ecor l and XhoI double enzymatic digestion
A: Recombinant plasmid PMD19-T-αB-crystallin; B: Recombinant plasmid pET28a-αB-crystallin. Lane 1: Marker; Lane 2: Target gene fragment.
Figure 2
Figure 2. Recombinant protein expression SDS-PAGE
Lane 1: Marker; Lane 2-5: 0.4 mmol/L and 0.6 mmol/L IPTG (supernatant and pellet).
Figure 3
Figure 3. Purification of the recombinant αB-crystallin by Q-Sepharose ion-exchange column.
Figure 4
Figure 4. Recombinant protein purified by chromatography
Lane 1-3: Simples, supernatant, pellet; Lane 4: Marker; Lane 5-9: p1, p2, p3, p4, p5 purified protein.
Figure 5
Figure 5. Coomassie Brilliant Blue staining (A) and Western blot analysis (B) of the recombinant protein
Lane 1: Marker; Lane 2: Recombinant human αB-crystallin.
Figure 6
Figure 6. Peptide mass fingerprinting analysis of the recombinant protein.
Figure 7
Figure 7. Identification of the recombinant protein by peptide mass fingerprint.
Figure 8
Figure 8. Insulin reduction assay
A: Negative group (Deionized water); B: Positive group (Insulin+DTT); C: Recombinant human αB-crystallin group (Insulin+DTT+αB-crystallin).
Figure 9
Figure 9. Recombinant human αB-crystallin molecular chaperone activity test.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Srivastava OP, Srivastava K. Existence of deamidated alphaB-crystallin fragments in normal and cataractous human lenses. Mol Vis. 2003;9:110–118. - PubMed
    1. Mao YW, Liu JP, Xiang H, Li DW. Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis. Cell Death Differ. 2004;11(5):512–526. - PubMed
    1. Sakaguchi H, Miyagi M, Darrow RM, Crabb JS, Hollyfield JG, Organisciak DT, Crabb JW. Intense light exposure changes the crystallin content in retina. Exp Eye Res. 2003;76(1):131–133. - PubMed
    1. Vázquez-Chona F, Song BK, Geisert EE., Jr Temporal changes in gene expression after injury in the rat retina. Invest Ophthalmol Vis Sci. 2004;45(8):2737–2746. - PMC - PubMed
    1. Kumar PA, Haseeb A, Suryanarayana P, Ehtesham NZ, Reddy GB. Elevated expression of alphaA- and alphaB-crystallins in streptozotocin-induced diabetic rat. Arch Biochem Biophys. 2005;15; 444(2):77–83. - PubMed

LinkOut - more resources