Cloning and Expression of LPS-Binding Single Domain Antibody Clones Selected from Phage Display Library of Indian Desert Camel

Authors

  • Ajit Singh Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125004 (Haryana), India
  • Akhil Kumar Gupta Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125004 (Haryana), India

DOI:

https://doi.org/10.37591/rrjoi.v4i1.1207

Abstract

Despite the absence of light chains in their structure, camelid and shark “heavy chain antibodies” (HCAbs) variable domains (designated as VHH) are able to make functional paratopes. The authors have selected lipopolysaccharide (LPS)-binder “single-domain antibodies” (dAb) clones from the previously constructed phage display library of (LPS)-immunized Indian desert camel. This study was undertaken to make available large amounts of some LPS-binder dAb clones by using a suitable expression vector-host system and convenient purification. Three Escherichia coli transformants producing LPS-binder dAb clones, designated as Cl16, Cl23 and Cl26 were amplified by VHH-PCR, inserted in pET 302/NT-His (Cl16) and pET 303/CT-His (Cl23 and Cl26) by directional cloning and transformed into chemi-competent BL21(DE3) E. coli host strain. The nucleotide sequencing was also done, the sequences were deposited in NCBI GenBank and assigned GenBank accession No. KF990216 (Cl23), KF990217 (Cl26) and KF990215 (Cl16). The dAb clones were expressed under IPTG induction at 37 °C for 10 h in a shaker incubator. The bacterial lysates were prepared for purification under denaturing conditions. The lysates and the purified products were analyzed by denaturing SDS-polyacrylamide gel electrophoresis. Examination of deduced amino acid sequences of these clones confirmed that they were derived from HCAbs. In SDS-PAGE, a strong band of about 17 kDa was visible in all the three clones. The clones could be purified by Nickel-chelate chromatography under denaturing conditions. The pET vector-BL21(DE) host proved to be an efficient system for expression of dAb clones.

 

Keywords: Single domain antibody, expression in pET vector-BL21(DE3) host,
LPS-binder dAb clones

 

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Published

2019-04-03

Issue

Vol. 4 No. 1 (2014)

Section

Research Article

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