Background: Previously, a single domain antibody clone (dAbCl26) selected from phage display library of LPS-immunized Indian desert camel was expressed in prokaryotic expression system and found to inhibit the LPS-induced inflammatory cytokine both in vitro and in vivo. However, the level of expression in prokaryotic expression system was low and also there was problem of endotoxin contamination. Pichia pastoris has emerged as an alternative host and economical approach for the expression of recombinant protein as it has the ability to properly refold the protein, gives high yield and produce protein without endotoxin contamination. Encouraged by the earlier findings of in vitro and in vivo inhibition by dAbCl26, the expression of dAbCl26 was investigated in yeast expression system P. pastoris. 
 
Methods: The dAbCl26 was cloned into the Pichia expression vector pPICZ&alpha;A and transformed into P. pastoris strain X33. Mutant phenotypes of selected clones were assessed using agar plate methods. For expression of the dAbCl26 in P. pastoris strain X33, the final growth conditions were optimized in buffered minimal methanol media with induction at OD600 4.0 and methanol induction at 0.5%. 
 
Result: Using the optimized conditions, a yield of about 4 mg/L of dAbCl26 was obtained in shake flask culture which is 2 fold higher than previously reported. The expressed protein was purified by using Ni-NTA chromatography and confirmed by Western blotting. The method developed in this study will be used as base for expression of other single domain antibodies and to scale up the expression on large scale. Based on its already reported functional properties, the Pichia expressed dAbCl26 can be a suitable candidate for further development as a therapeutic agent for endotoxaemia in animals.

Optimization of Culture Conditions for Expression of Anti-lipopolysaccharide Single Domain Antibody Clone in Pichia pastoris

Regucalcin is a multifunctional protein having an important role in calcium homeostasis, L-ascorbic acid biosynthesis, anti-prolific, anti-apoptotic functions as well as detoxification of chemical warfare nerve agents. &nbsp;Recently, it has been localized to male reproductive tract of rat and human, and identified as an androgen-target gene. The literature suggests a possible role of regucalcin in male fertility. However, no detailed studies have been conducted on its role in male reproductive organs of domestic animals. As an initial step, we had cloned and expressed regucalcin in Pichia pastoris. The sequence analysis showed 100% homology with regucalcin of Bos tours both at nucleotide and amino acid level. The SDS-PAGE and Western blot studies of recombinant protein probed with anti-regucalcin monoclonal antibody showed a higher molecular weight (56 kDa) than the expected (35.5 kDa) that could be due to hyperglycosylation. The recombinant regucalcin and its antibodies can be used to study the detailed role of the protein in male reproduction.

Optimization of Culture Conditions for Expression of Anti-lipopolysaccharide Single Domain Antibody Clone in Pichia pastoris

Expression of the secretory form of recombinant regucalcin of Bubalus bubalis in methylotropic yeast