Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non-aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross-linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).

http://largens.com/biochemlab/peroxidase2.pdf

Enzyme immobilization: The quest for optimum performance

Production of recombinant proteins by microbes and higher organisms.

Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris.

Recombinant protein expression for therapeutic applications.

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Production of these proteins has a remarkable demand in the market. Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically therapeutic proteins such as insulin and bovine growth hormone. These demands have driven the development of a variety of strategies for achieving high-level expression of protein, particularly involving several aspects such as expression vectors design, gene dosage, promoter strength (transcriptional regulation), mRNA stability, translation initiation and termination (translational regulation), host design considerations, codon usage, and fermentation factors available for manipulating the expression conditions, which are the major challenges is obtaining the high yield of protein at low cost.

Strategies for efficient production of heterologous proteins in Escherichia coli.

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form.

Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product.

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterolo- gous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expres- sion product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, hu- man insulin with full receptor binding capacity and bio- logical activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form. © 2001 John Wiley & Sons, Inc. Biotech- nol Bioeng 73: 74-79, 2001.

COMMUNICATION TO THE EDITOR Human Insulin from a Precursor Overexpressed in the Methylotrophic Yeast Pichia pastoris and a Simple Procedure for Purifying the Expression Product

A synthetic single-chain porcine insulin precursor (PIP) gene and an alpha-mating factor leader sequence (alpha MFL) gene obtained by the PCR method are inserted between the promoter and 3'-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/alpha MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cerevisiae transformed by pVT102-U/alpha MFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.

Secretory expression of a single-chain insulin precursor in yeast and its conversion into human insulin

Blood glucose lowering assay proved that [B16Ala]insulin and [B26Ala]insulin exhibit potency of acute blood glucose lowering in normal pigs, which demonstrates that they are fast-acting insulin. Single-chain precursor of [B16Ala]insulin and [B26Ala]insulin is [B16Ala]PIP and [B26Ala]PIP, respectively, which are suitable for gene expression. Secretory expression level of the precursors in methylotrophic yeast Pichia pastoris was quite high, 650 mg/L and 130 mg/L, respectively. In vivo biological assay showed that the two fast-acting insulins have full or nearly full biological activity. So both [B16Ala]insulin and [B26Ala]insulin can be well developed as fast-acting insulin for clinic use.

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

Aim: To explore the importance of B9 and B10 amino acid residues in the insulin molecule.
 Methods: The [B9Glu, B10Asp] insulin (E,D-insulin) receptor binding activity, glucose upta ke activity, and lipogenesis activity were measured in isolated adipocytes. The translocation of glucose transporter 4 (Glut4) and the phosphorylation of insulin receptor (IR) stimulated by E, D-insulin were determined by Western blotting.
 Results: Compared with native insulin, the receptor binding activity of E,D-insulin was 31 %; the stimulating activity of E,D-insulin in glucose transport and lipogenesis were 45 % and 40 % respectively; the stimulations of Glut4 translocation and insulin receptor autophosphorylation of E,D-insulin were about 58 % and 46 % respectively.
 Conclusion: B9-serine residue is crucial for insulin actions in glucose metabolism.

Yue-Hua Tang

Papers

Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product.

COMMUNICATION TO THE EDITOR Human Insulin from a Precursor Overexpressed in the Methylotrophic Yeast Pichia pastoris and a Simple Procedure for Purifying the Expression Product

Secretory expression of a single-chain insulin precursor in yeast and its conversion into human insulin

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

B9-serine residue is crucial for insulin actions in glucose metabolism