Periplasmic secretion of human growth hormone by <i>Escherichia coli</i>

Chang, Judy Y. H.; Pai, Rong-Chang; Bennett, William F.; Bochner, Barry R.

doi:10.1042/bst0170335

Cited by 16 publications

(11 citation statements)

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“…Upon reduction the 25 kDa species migrates as two low molecular weight fragments suggesting that the 25 kDa species is the result of a previously characterized endoproteolytic cleavage between Thr-142 and Tyr-143 of hGH during culture [25]. This “two chain” hGH is commonly observed in periplasmic preparations and has equivalent biological activity as non-cleaved hGH in both the hyphopysectomized rat weight-gain and Nb2 cell growth assay [21, 25, 30]. Further optimization of culture conditions such as pH, temperature, cation concentration, etc.…”

Section: Resultsmentioning

confidence: 99%

Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

Sockolosky

Szoka

2013

Protein Expression and Purification

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A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (DE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni2+ affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli.

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Section: Resultsmentioning

confidence: 99%

Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

Sockolosky

Szoka

2013

Protein Expression and Purification

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“…hGH was expressed and purified as described previously (Chang et al 1987). A truncated form (residues 29-238) of the ECD of the hGHR was expressed and purified as described (Fuh et al 1990;Clackson et al 1998), except the receptor was eluted with 4.5 M MgCl 2 off a hGH affinity column.…”

Section: Sample Preparationmentioning

confidence: 99%

Site2 binding energetics of the regulatory step of growth hormone–induced receptor homodimerization

et al. 2003

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Receptor signaling in the growth hormone (GH)-growth hormone receptor (GHR) system is controlled through a sequential two-step hormone-induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of the second ECD (ECD2) to the stable preassociated 1 : 1 GH/ECD1 complex on the cell surface. To determine the energetics that governs this step, the binding kinetics of 38 single-and double-alanine mutants in the hGH Site2 contact with ECD2 were measured by using trimolecular surface plasmon resonance (TM-SPR). We find that the Site2 interface of hGH does not have a distinct binding hot-spot region, and the most important residues are not spatially clustered, but rather are distributed over the whole binding surface. In addition, it was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues. Residues that show little effect or even improved binding on substitution with alanine, when paired with D116A-hGH, display significant negative cooperativity. Because most of these pairwise mutated residues are spatially separated by Ն10 Å, this indicates that the Site2 binding interface of the hGH-hGHR ternary complex displays both structural and energetic malleability.

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“…Potentially higher volume needs, due to newer indications (Mehta and Hindmarsh, 2002) and alternative delivery systems (Lee et al, 1997;Leone-Bay et al, 1996), have led to the investigation of hGH production in other hosts, including other bacteria (Franchi et al, 1991), fungi (Ecamilla-Treviño et al, 2000), milk from transgenic mammals (Lubon and Palmer, 2000), and tobacco plants (Leite et al, 2000;Staub et al, 2000). However, most hGH biochemical characterization data are from humans (Baumann, 1991) or E. coli (Chang et al, 1987;Chang et al, 1989), typically described as a single chain of 191 amino acids, an N-terminus of Phe-Pro-Thr formed after removal of the signal peptide during secretion, and two intrachain disulfide bonds. Natural circulating variants include dimers, internal truncations from alternative splicing, protein cleavage products, N-terminal acylation, and deamidations (Chang et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

Host limits to accurate human growth hormone production in multiple plant systems

Russell

Spatola

Dian

et al. 2005

Biotech & Bioengineering

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Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.

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Periplasmic secretion of human growth hormone by Escherichia coli

Cited by 16 publications

References 1 publication

Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

Site2 binding energetics of the regulatory step of growth hormone–induced receptor homodimerization

Host limits to accurate human growth hormone production in multiple plant systems

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