Molecular cloning and expression of a putative crustacean hyperglycemic hormone of Litopenaeus vannamei in Pichia pastoris

Abstract

Crustacean hyperglycemic hormone (CHH) is the most abundant and best studied member of the CHH/MIH/GIH neuropeptide hormone family. CHH plays a major role in controlling glucose levels in the hemolymph, and it also has significance in regulating molting, reproduction, and osmoregulation. In contrast, molt-inhibiting hormone (MIH) is responsible for maintaining animals in an intermolt stage. In this study, Liv-MIH-1 cDNA, which encodes a mature neuropeptide from the eyestalk of white shrimp, Litopenaeus vannamei, was expressed in methylotrophic yeast (Pichia pastoris KM71) under the control of an alcohol oxidase promoter. Recombinant Liv-MIH-1 was *Corresponding author secreted into the culture medium using the α-factor prepro-sequence without Glu-Ala repeats. The expected protein, which had an apparent molecular mass of 12.1 kDa, was detected by Tricine-SDS-PAGE analysis and confirmed by Western blot. Pure recombinant Liv-MIH-1 was obtained by affinity chromatography, and N-terminal sequence analysis confirmed expression of the protein. Biological assays for CHH and MIH activity were also performed. Purified recombinant Liv-MIH-1 showed the ability to elevate the glucose level of hemolymph of L. vannamei, but molting was unaffected. Since these results are in agreement with the high structural and phylogenetic similarity that has been

Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei

“…Interestingly, we found that CHH-B1 has hyperglycemic activity even though the sequence of the native CHH-B1 suggests that it has a free C-terminus. Our present results confirmed the hyperglycemic activity that was previously observed using a CHH-B1 recombinant version having 26 additional amino acids at the C-terminal end, constructed previously by our group using unilaterally ablated shrimp [30].…”

“…The expression of rCHH-B1 was induced according to the method described by Sánchez-Castrejón et al [30] with some modifications. A colony of P. pastoris X-33 containing pPicZ␣A-CHH-B1a inserted into its genome was grown in 3 mL of YPD medium (1% yeast extract, 2% peptone, 2% dextrose, 100 g/mL zeocin) for 18 h at 30 • C and 200 rpm.…”

“…Bands of ∼10 kDa were not detected in control samples. Since the Western blot analysis of culture supernatants showed that the expression of rCHH-B1 was similar among the induction conditions analyzed, the condition of 2% methanol for 24 h was selected [38]; Pt, P. trituberculatus (CHH-1, ACB46189; CHH-2, AIZ94611) [49]; Cs, C. sapidus (ES-CHH (CHH-1), AAS45136; CsPO-CHH (CsCHH-2, ABG67921, ABC61678) [8,18,53]; Gel, G. lateralis (EG-CHH-A, ABF48652; Po-CHH, ABF58091) [22,52]; Cam, C. maenas (SG-CHH, AAG29442; PO-CHH, AAG29435) [10]; Sco, S. olivacea (CHH, AAQ75760; CHH-L, ABP88270) [2,41]; Poi, P. ibericum (CHHXO, ABA70560; CHHPO, ABA70561) [38]; Mar, M. rosenbergii (CHH, AAF29534; CHH-L, AF372657) [3,29]; Prc, P. clarkii (CHH1, BAA89003; CHH1-L, AAL79193; CHH2, AFV95082; CHH2-L, AFV95078) [19,48]; Pj, P. japonica (CHH1ES, AFG16933; CHH1PO, AFG16932) [16]; Lv, L. vannamei (CHH-B2, AAN86057; CHH-B1, AY167045) [21,30]; Hyperglycemic activity tested in vivo: N, using native neuropeptides; R, recombinant neuropeptides; (+), with activity; (−) without activity; aa, number of amino acids in sequences; % I.D., identity percentage relative to the CHH-B1 sequence. CHH, CHH isoforms; CHH-L, CHH-like isoforms.…”

“…Even though the recombinant peptide showed hyperglycemic activity in vivo, it did not show molt-inhibiting activity [30]. In the present work, we report the cloning in P. pastoris, expression and purification of the L. vannamei recombinant CHH-B1 isoform with a free C-terminus (rCHH-B1) and identical to the native neurohormone, as well as an examination of its hyperglycemic activity using a dose-response bioassay with bilaterally ablated shrimp.…”

A chimeric recombinant crustacean hyperglycemic hormone fromLitopenaeus schmitti(Burkenroad) obtained as C-terminus fusion protein boost hemolymph glucose concentration inLitopenaeus vannamei(Boone)

“…In addition, the use of the DNA recombinant technology has been useful in generating these crustacean peptides in sufficient quantities to enable furthermore studies of their physiological roles (Okumura 2004). Until now, the recombinant CHHs (rCHHs) have been obtained by bacterial (Gu, Yu & Chan 2000;Katayama, Ohira, Aida & Nagasawa 2002;Mettulio, Edomi, Ferrero, Lorenzon & Giulianini 2004a;Ohira, Tsutsui, Nagasawa & Wilder 2006;Mosco, Edomi, Guarnaccia, Lorenzon, Pongor, Ferrero & Giulianini 2008) or yeast expression systems (Ohira, Katayama, Aida & Nagasawa 2003;Udomkit, Treerattrakool & Panyim 2004;Sánchez-Castrejón, Ponce-Rivas, Aguilar & Díaz 2008) but the majority of the rCHH proteins expressed in Escherichia coli has been obtained as insoluble aggregates (Gu et al 2000;Katayama et al 2002;Ohira et al 2006;Mosco et al 2008). On the other hand, although the CHH protein has been expressed in a soluble form and biologically active in the yeast expression system, it has been shown that their yield is much less than that obtained in E. coli (Udomkit et al 2004).…”